Plant RNaseD-like genes

ABSTRACT

This invention relates to an isolated nucleic acid fragment encoding a RNaseD-like protein. The invention also relates to the construction of a chimeric gene encoding all or a substantial portion of the RNaseD-like protein, in sense or antisense orientation, wherein expression of the chimeric gene results in production of altered levels of the RNaseD-like protein in a transformed host cell.

[0001] This application claims the benefit of U.S. ProvisionalApplication No. 60/218993, filed Jul. 17, 2000, the entire contents ofwhich are herein incorporated by reference.

FIELD OF THE INVENTION

[0002] This invention is in the field of plant molecular biology. Morespecifically, this invention pertains to nucleic acid fragments encodingRNaseD-like proteins in plants and seeds.

BACKGROUND OF THE INVENTION

[0003] In genetically modified plants transgenes can cause the silencingof endogenous plant genes if they are sufficiently homologous. Thisphenomenon is known as co-suppression. The exact mechanism ofco-suppression in plants is unknown, however several factors such astransgene copy number, sequence similarity and degree of repetitivenessbetween the transgene and endogenous gene(s), and transgene expressionlevels appear to play a role (Stam et al. (1997) Annals of Botany79:3-13). The phenomenon of co-supresson by transgenic DNA has also beenobserved in many organisms from fungi to animals. Most recently themut-7 gene (a homolog of the RNaseD gene) of C. elegans has beenimplicated in the co-suppression mechanism in that organism (Hammond, S.et al. (2000) Nature 404:293-296), (Ketting R. F. et al. (1999) Cell99(2): 133-141).

[0004] There is a great deal of interest in identifying the genes thatencode proteins like RNaseD that may be involved in co-suppressionmechanisms in plants. RNaseD has three catalytic domains that arediagnostic of the protein and each of the plant sequences presentedbelow have conserved amino acid residues in these domains suggesting thegenes encode RNaseD-like proteins. The genes that code for RNaseD-likeproteins may be used to further study co-suppression and developspecific methods to regulate gene expression via co-suppressionmethodology. Accordingly, the availability of nucleic acid sequencesencoding all or a substantial portion of RNaseD molecules wouldfacilitate studies to better understand co-suppression and providegenetic tools and methods to manipulate gene expression in plants.

SUMMARY OF THE INVENTION

[0005] The present invention concerns an isolated polynucleotidecomprising (a) a nucleotide sequence encoding a polypeptide comprisingat least 133 amino acids wherein the amino acid sequence of thepolypeptide and the amino acid sequence of SEQ ID NO: 2, 4, 6, 8, 10,12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46,or 48 have at least 50%, 55%, 60%, 65%, 70%, 75%, 80%,.85%, 90%, or 95%sequence identity based on the Clustal alignment method, or (b) thecomplement of the nucleotide sequence, wherein the complement containsthe same number of nucleotides as the nucleotide sequence and is 100%complementary to the nucleotide sequence. The polypeptide preferablycomprises the amino acid sequence of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14,16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, or 48.The nucleotide sequence preferably comprises the nucleotide sequence ofSEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31,33, 35, 37, 39, 41, 43, 45, or 47. The polypeptide preferably has theactivity of a ribonuclease D (RNaseD).

[0006] In a second embodiment, the present invention relates to achimeric gene comprising any of the isolated polynucleotides of thepresent invention operably linked to a regulatory sequence, and a cell,a plant, and a seed comprising the chimeric gene.

[0007] In a third embodiment, the present invention relates to a vectorcomprising any of the isolated polynucleotides of the present invention.

[0008] In a fourth embodiment, the present invention relates to anisolated polynucleotide fragment comprising a nucleotide sequencecomprised by any of the polynucleotides of the present invention,wherein the nucleotide sequence contains at least 30, 40, or 60nucleotides.

[0009] In a fifth embodiment, the present invention relates to a methodfor transforming a cell comprising transforming a cell with any of theisolated polynucleotides of the present invention, and the celltransformed by this method. Advantageously, the cell is eukaryotic,e.g., a yeast or plant cell, or prokaryotic, e.g., a bacterium.

[0010] In a sixth embodiment, the present invention relates to a methodfor producing a transgenic plant comprising transforming a plant cellwith any of the isolated polynucleotides of the present invention andregenerating a plant from the transformed plant cell, the transgenicplant produced by this method, and the seed obtained from the transgenicplant.

[0011] In a seventh embodiment, the present invention concerns anisolated polypeptide comprising an amino acid sequence comprising atleast 133 amino acids, wherein the amino acid sequence and the aminoacid sequence of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24,26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, or 48, have at least 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% sequence identity basedon the Clustal alignment method. The amino acid sequence preferablycomprises the amino acid sequence of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14,16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, or 48.The polypeptide preferably has RnaseD activity.

[0012] In an eighth embodiment, the present invention relates to avirus, preferably a baculovirus, comprising any of the isolatedpolynucleotides of the present invention or any of the chimeric genes ofthe present invention.

[0013] In a ninth embodiment, the invention relates to a method ofselecting an isolated polynucleotide that affects the level ofexpression of a RNaseD or its enzyme activity in a host cell, preferablya plant cell, the method comprising the steps of: (a) constructing anisolated polynucleotide of the present invention or an isolated chimericgene of the present invention; (b) introducing the isolatedpolynucleotide or the isolated chimeric gene into a host cell; (c)measuring the level of the RNaseD or its enzyme activity in the hostcell containing the isolated polynucleotide; and (d) comparing the levelof the RNaseD or its enzyme activity in the host cell containing theisolated polynucleotide with the level of the RNaseD or its enzymeactivity in the host cell that does not contain the isolatedpolynucleotide.

[0014] In a tenth embodiment, the invention concerns a method ofobtaining a nucleic acid fragment encoding a substantial portion of aRNaseD, preferably a plant RNaseD, comprising the steps of synthesizingan oligonucleotide primer comprising a nucleotide sequence of at leastone of 30 (preferably at least one of 40, most preferably at least oneof 60) contiguous nucleotides derived from a nucleotide sequenceselected from the group consisting of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13,15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, or 47,and the complement of such nucleotide sequences; and amplifying anucleic acid fragment (preferably a cDNA inserted in a cloning vector)using the oligonucleotide primer. The amplified nucleic acid fragmentpreferably will encode a substantial portion of the amino acid sequenceof a RNaseD.

[0015] In an eleventh embodiment, this invention relates to a method ofobtaining a nucleic acid fragment encoding all or a substantial portionof the amino acid sequence encoding a RNaseD comprising the steps of:probing a cDNA or genomic library with an isolated polynucleotide of thepresent invention; identifying a DNA clone that hybridizes with anisolated polynucleotide of the present invention; isolating theidentified DNA clone; and sequencing the cDNA or genomic fragment thatcomprises the isolated DNA clone.

[0016] In a twelfth embodiment, this invention concerns a method forpositive selection of a transformed cell comprising: (a) transforming ahost cell with the chimeric gene of the present invention or anexpression cassette of the present invention; and (b) growing thetransformed host cell, preferably a plant cell, such as a monocot or adicot, under conditions which allow expression of the polynucleotideencoding a RNaseD in an amount sufficient to complement a null mutant toprovide a positive selection means.

[0017] In a thirteenth embodiment, this invention relates to a method ofaltering the level of expression of a RNaseD in a host cell comprising:(a) transforming a host cell with a chimeric gene of the presentinvention; and (b) growing the transformed host cell under conditionsthat are suitable for expression of the chimeric gene wherein expressionof the chimeric gene results in production of altered levels of theRNaseD in the transformed host cell.

[0018] In a fourteenth embodiment, this invention relates to a methodfor evaluating at least one compound for its ability to inhibit theactivity of a RNaseD, the method comprising the steps of: (a)transforming a host cell with a chimeric gene comprising a nucleic acidfragment encoding a RNaseD polypeptide, operably linked to suitableregulatory sequences; (b) growing the transformed host cell underconditions that are suitable for expression of the chimeric gene whereinexpression of the chimeric gene results in production of RNaseDpolypeptide in the transformed host cell; (c) optionally purifying theRNaseD polypeptide expressed by the transformed host cell; (d) treatingthe RNaseD polypeptide with a compound to be tested; and (e) comparingthe activity of the RNaseD polypeptide that has been treated with a testcompound to the activity of an untreated RNaseD polypeptide, andselecting compounds with potential for inhibitory activity.

BRIEF DESCRIPTION OF THE SEQUENCE LISTINGS

[0019] The invention can be more fully understood from the followingdetailed description and the accompanying Sequence Listing which form apart of this application.

[0020] Table 1 lists the polypeptides that are described herein, thedesignation of the cDNA clones that comprise the nucleic acid fragmentsencoding polypeptides representing all or a substantial portion of thesepolypeptides, and the corresponding identifier (SEQ ID NO:) as used inthe attached Sequence Listing. The sequence descriptions and SequenceListing attached hereto comply with the rules governing nucleotideand/or amino acid sequence disclosures in patent applications as setforth in 37 C.F.R. §1.821-1.825. TABLE 1 RNaseD-like Proteins SEQ ID NO:(Nucleo- (Amino Protein Clone Designation tide) Acid) maize [Zea mays]cco1n.pk063.p21 1 2 maize [Zea mays] contig of: 3 4 cco1n.pk058.p8,cco1n.pk094.o1, p0010.cbpbx36r, p0098.cdfao60r maize [Zea mays]p0018.chstb24r 5 6 maize [Zea mays] p0046.cndai04r 7 8 maize [Zea mays]p0133.ctvad13r 9 10 rice [Oryza sativa] rcaln.pk023.m3 11 12 soybean[Glycine max] scr1c.pk004.m6 13 14 soybean [Glycine max] src3c.pk013.c1215 16 vernonia [Vernonia vs1.pk0011.b11 17 18 vespilifolia] wheat-common[Triticum w1mk8.pk0027.f7 19 20 aestivum] wheat-common [Triticumwne1g.pk005.e5 21 22 aestivum] maize [Zea mays] cco1n.pk063.p21:fis 2324 maize [Zea mays] cepe7.pk0008.b7:fis 25 26 maize [Zea mays] contigof: 27 28 p0046.cndai04r:fis, p0128.cpicn88r wheat-common [Triticumw1mk8.pk0022.f7:fis 29 30 aestivum] columbine [Aquilegiaeav1c.pk005.f18:fis 31 32 vulgaris] grape [Vitis sp.] vmb1c.pk010.17:fis33 34 maize [Zea mays] csi1n.pk0017.f10:fis 35 36 para rubber [Heveaehb2c.pk006.p12:fis 37 38 brasiliensis] rice [Oryza sativa]rdi2c.pk011.f3:fis 39 40 soybean [Glycine max] scn1c.pk001.p19:fis 41 42soybean [Glycine max] sea1c.pk015.i18 43 44 soybean [Glycine max]sgs1c.pk003.h10 45 46 sunflower [Helianthus sp.] hss1c.pk015.a23 47 48

[0021] The Sequence Listing contains the one letter code for nucleotidesequence characters and the three letter codes for amino acids asdefined in conformity with the IUPAC-IUBMB standards described inNucleic Acids Res. 13:3021-3030 (1985) and in the Biochemical J. 219(No. 2):345-373 (1984) which are herein incorporated by reference. Thesymbols and format used for nucleotide and amino acid sequence datacomply with the rules set forth in 37 C.F.R. §1.822.

[0022] Conserved sequence domains within the polypeptide sequences areuseful in identifying RNaseD. Such domains can include, but are notlimited to, Domain I: vgxDxEwxp; Domain II: gxxxxxD; and Domain III:wxxxplxxxqxxYaa,D; where the underlined amino acids are conservedresidues, the capital amino acids are acidic residues, and x representsany amino acid; and the separation between domains can be anywhere from43 to 85 amino acids.

[0023] The nucleotide sequences of SEQ ID NOs: 1, 5, and 15 (clonescco1n.pk063.p21, p0018.chstb24r, and src3c.pk013.cl2, respectively) arebelieved to be full length based upon experiments designed to isolatethe 5′-end of the cognate endogenous mRNA. The polypeptide encoded bySEQ ID NO: 1 is only 3 amino acids short of the full length enzyme(shown in the full insert sequences of SEQ ID NOs: 23 and 24). The fullinsert sequence of a second clone, cepe7.pk0008.b7:fis (SEQ ID NO: 25),confirms the sequence found in SEQ ID NO: 5. Therefore, the polypeptidefound in SEQ ID NO: 6 represents the complete enzyme. The polypeptideencoded by SEQ ID NO: 15 most likely initiates with the methionine atposition 77 of SEQ ID NO: 16.

[0024] Other clones that represent complete (or nearly complete) genesequences include eav1c.pk005.f1 8:fis (SEQ ID NOs: 31 and 32),csi1n.pk0017.f10:fis (SEQ ID NOs: 35 and 36), contig ofp0046.cndai04r:fis and p0128.cpicn88r (SEQ ID NOs: 27 and 28),ehb2c.pk006.pl2:fis (SEQ ID NOs: 37 and 38), rdi2c.pk011.f3:fis (SEQ IDNOs: 39 and 40), scn1c.pk001.p19:fis (SEQ ID NOs: 41 and 42), andhss1c.pk015.a23 (SEQ ID NOs: 47 and 48).

DETAILED DESCRIPTION OF THE INVENTION

[0025] In the context of this disclosure, a number of terms shall beutilized. The terms “polynucleotide”, “polynucleotide sequence”,“nucleic acid sequence”, and “nucleic acid fragment”/“isolated nucleicacid fragment” are used interchangeably herein. These terms encompassnucleotide sequences and the like. A polynucleotide may be a polymer ofRNA or DNA that is single- or double-stranded, that optionally containssynthetic, non-natural or altered nucleotide bases. A polynucleotide inthe form of a polymer of DNA may be comprised of one or more segments ofcDNA, genomic DNA, synthetic DNA, or mixtures thereof. An isolatedpolynucleotide of the present invention may include at least one of 30contiguous nucleotides, preferably at least one of 40 contiguousnucleotides, most preferably one of at least 60 contiguous nucleotidesderived from SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25,27, 29, 31, 33, 35, 37, 39, 41, 43, 45,or 47,or the complement of suchsequences.

[0026] The term “isolated” refers to materials, such as nucleic acidmolecules and/or proteins, which are substantially free or otherwiseremoved from components that normally accompany or interact with thematerials in a naturally occurring environment. Isolated polynucleotidesmay be purified from a host cell in which they naturally occur.Conventional nucleic acid purification methods known to skilled artisansmay be used to obtain isolated polynucleotides. The term also embracesrecombinant polynucleotides and chemically synthesized polynucleotides.

[0027] The term “recombinant” means, for example, that a nucleic acidsequence is made by an artificial combination of two otherwise separatedsegments of sequence, e.g., by chemical synthesis or by the manipulationof isolated nucleic acids by genetic engineering techniques.

[0028] As used herein, “contig” refers to a nucleotide sequence that isassembled from two or more constituent nucleotide sequences that sharecommon or overlapping regions of sequence homology. For example, thenucleotide sequences of two or more nucleic acid fragments can becompared and aligned in order to identify common or overlappingsequences. Where common or overlapping sequences exist between two ormore nucleic acid fragments, the sequences (and thus their correspondingnucleic acid fragments) can be assembled into a single contiguousnucleotide sequence.

[0029] As used herein, “substantially similar” refers to nucleic acidfragments wherein changes in one or more nucleotide bases results insubstitution of one or more amino acids, but do not affect thefunctional properties of the polypeptide encoded by the nucleotidesequence. “Substantially similar” also refers to nucleic acid fragmentswherein changes in one or more nucleotide bases does not affect theability of the nucleic acid fragment to mediate alteration of geneexpression by gene silencing through for example antisense orco-suppression technology. “Substantially similar” also refers tomodifications of the nucleic acid fragments of the instant inventionsuch as deletion or insertion of one or more nucleotides that do notsubstantially affect the functional properties of the resultingtranscript vis-à-vis the ability to mediate gene silencing or alterationof the functional properties of the resulting protein molecule. It istherefore understood that the invention encompasses more than thespecific exemplary nucleotide or amino acid sequences and includesfunctional equivalents thereof. The terms “substantially similar” and“corresponding substantially” are used interchangeably herein.

[0030] Substantially similar nucleic acid fragments may be selected byscreening nucleic acid fragments representing subfragments ormodifications of the nucleic acid fragments of the instant invention,wherein one or more nucleotides are substituted, deleted and/orinserted, for their ability to affect the level of the polypeptideencoded by the unmodified nucleic acid fragment in a plant or plantcell. For example, a substantially similar nucleic acid fragmentrepresenting at least one of 30 contiguous nucleotides derived from theinstant nucleic acid fragment can be constructed and introduced into aplant or plant cell. The level of the polypeptide encoded by theunmodified nucleic acid fragment present in a plant or plant cellexposed to the substantially similar nucleic fragment can then becompared to the level of the polypeptide in a plant or plant cell thatis not exposed to the substantially similar nucleic acid fragment.

[0031] For example, it is well known in the art that antisensesuppression and co-suppression of gene expression may be accomplishedusing nucleic acid fragments representing less than the entire codingregion of a gene, and by using nucleic acid fragments that do not share100% identity with the gene to be suppressed. Moreover, alterations in anucleic acid fragment which result in the production of a chemicallyequivalent amino acid at a given site, but do not effect the functionalproperties of the encoded polypeptide, are well known in the art. Thus,a codon for the amino acid alanine, a hydrophobic amino acid, may besubstituted by a codon encoding another less hydrophobic residue, suchas glycine, or a more hydrophobic residue, such as valine, leucine, orisoleucine. Similarly, changes which result in substitution of onenegatively charged residue for another, such as aspartic acid forglutamic acid, or one positively charged residue for another, such aslysine for arginine, can also be expected to produce a functionallyequivalent product. Nucleotide changes which result in alteration of theN-terminal and C-terminal portions of the polypeptide molecule wouldalso not be expected to alter the activity of the polypeptide. Each ofthe proposed modifications is well within the routine skill in the art,as is determination of retention of biological activity of the encodedproducts. Consequently, an isolated polynucleotide comprising anucleotide sequence of at least one of 30 (preferably at least one of40, most preferably at least one of 60) contiguous nucleotides derivedfrom a nucleotide sequence selected from the group consisting of SEQ IDNO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35,37, 39, 41, 43, 45, or 47, and the complement of such nucleotidesequences may be used in methods of selecting an isolated polynucleotidethat affects the expression of a RNaseD-like polypeptide in a host cell.A method of selecting an isolated polynucleotide that affects the levelof expression of a polypeptide in a virus or in a host cell (eukaryotic,such as plant or yeast, prokaryotic such as bacterial) may comprise thesteps of: constructing an isolated polynucleotide of the presentinvention or a chimeric gene of the present invention; introducing theisolated polynucleotide or the chimeric gene into a host cell; measuringthe level of a polypeptide or enzyme activity in the host cellcontaining the isolated polynucleotide; and comparing the level of apolypeptide or enzyme activity in the host cell containing the isolatedpolynucleotide with the level of a polypeptide or enzyme activity in ahost cell that does not contain the isolated polynucleotide.

[0032] Moreover, substantially similar nucleic acid fragments may alsobe characterized by their ability to hybridize. Estimates of suchhomology are provided by either DNA-DNA or DNA-RNA hybridization underconditions of stringency as is well understood by those skilled in theart (Hames and Higgins, Eds. (1985) Nucleic Acid Hybridisation, IRLPress, Oxford, U.K.). Stringency conditions can be adjusted to screenfor moderately similar fragments, such as homologous sequences fromdistantly related organisms, to highly similar fragments, such as genesthat duplicate functional enzymes from closely related organisms.Post-hybridization washes determine stringency conditions. One set ofpreferred conditions uses a series of washes starting with 6X SSC, 0.5%SDS at room temperature for 15 min, then repeated with 2X SSC, 0.5% SDSat 45° C. for 30 min, and then repeated twice with 0.2X SSC, 0.5% SDS at50° C. for 30 min. A more preferred set of stringent conditions useshigher temperatures in which the washes are identical to those aboveexcept for the temperature of the final two 30 min washes in 0.2X SSC,0.5% SDS which was increased to 60° C. Another preferred set of highlystringent conditions uses two final washes in 0.1X SSC, 0.1% SDS at 65°C.

[0033] Substantially similar nucleic acid fragments of the instantinvention may also be characterized by the percent identity of the aminoacid sequences that they encode to the amino acid sequences disclosedherein, as determined by algorithms commonly employed by those skilledin this art. Suitable nucleic acid fragments (isolated polynucleotidesof the present invention) encode polypeptides that are at least about70% identical, preferably at least about 80% identical to the amino acidsequences reported herein. Preferred nucleic acid fragments encode aminoacid sequences that are about 85% identical to the amino acid sequencesreported herein. More preferred nucleic acid fragments encode amino acidsequences that are at least about 90% identical to the amino acidsequences reported herein. Most preferred are nucleic acid fragmentsthat encode amino acid sequences that are at least about 95% identicalto the amino acid sequences reported herein. Suitable nucleic acidfragments not only have the above identities but typically encode apolypeptide having at least 50 amino acids, preferably at least 100amino acids, more preferably at least 150 amino acids, still morepreferably at least 200 amino acids, and most preferably at least 250amino acids. Sequence alignments and percent identity calculations wereperformed using the Megalign program of the LASERGENE bioinformaticscomputing suite (DNASTAR Inc., Madison, Wis.). Multiple alignment of thesequences was performed using the Clustal method of alignment (Higginsand Sharp (1989) CABIOS. 5:151-153) with the default parameters (GAPPENALTY=10, GAP LENGTH PENALTY=10). Default parameters for pairwisealignments using the Clustal method were KTUPLE 1, GAP PENALTY=3,WINDOW=5 and DIAGONALS SAVED=5.

[0034] A “substantial portion” of an amino acid or nucleotide sequencecomprises an amino acid or a nucleotide sequence that is sufficient toafford putative identification of the protein or gene that the aminoacid or nucleotide sequence comprises. Amino acid and nucleotidesequences can be evaluated either manually by one skilled in the art, orby using computer-based sequence comparison and identification toolsthat employ algorithms such as BLAST (Basic Local Alignment Search Tool;Altschul et al. (1993) J. Mol. Biol. 215:403-410; see alsowww.ncbi.nlm.nih.gov/BLAST/). In general, a sequence of ten or morecontiguous amino acids or thirty or more contiguous nucleotides isnecessary in order to putatively identify a polypeptide or nucleic acidsequence as homologous to a known protein or gene. Moreover, withrespect to nucleotide sequences, gene-specific oligonucleotide probescomprising 30 or more contiguous nucleotides may be used insequence-dependent methods of gene identification (e.g., Southernhybridization) and isolation (e.g., in situ hybridization of bacterialcolonies or bacteriophage plaques). In addition, short oligonucleotidesof 12 or more nucleotides may be used as amplification primers in PCR inorder to obtain a particular nucleic acid fragment comprising theprimers. Accordingly, a “substantial portion” of a nucleotide sequencecomprises a nucleotide sequence that will afford specific identificationand/or isolation of a nucleic acid fragment comprising the sequence. Theinstant specification teaches amino acid and nucleotide sequencesencoding polypeptides that comprise one or more particular plantproteins. The skilled artisan, having the benefit of the sequences asreported herein, may now use all or a substantial portion of thedisclosed sequences for purposes known to those skilled in this art.Accordingly, the instant invention comprises the complete sequences asreported in the accompanying Sequence Listing, as well as substantialportions of those sequences as defined above.

[0035] “Codon degeneracy” refers to divergence in the genetic codepermitting variation of the nucleotide sequence without effecting theamino acid sequence of an encoded polypeptide. Accordingly, the instantinvention relates to any nucleic acid fragment comprising a nucleotidesequence that encodes all or a substantial portion of the amino acidsequences set forth herein. The skilled artisan is well aware of the“codon bias” exhibited by a specific host cell in usage of nucleotidecodons to specify a given amino acid. Therefore, when synthesizing anucleic acid fragment for improved expression in a host cell, it isdesirable to design the nucleic acid fragment such that its frequency ofcodon usage approaches the frequency of preferred codon usage of thehost cell.

[0036] “Synthetic nucleic acid fragments” can be assembled fromoligonucleotide building blocks that are chemically synthesized usingprocedures known to those skilled in the art. These building blocks areligated and annealed to form larger nucleic acid fragments which maythen be enzymatically assembled to construct the entire desired nucleicacid fragment. “Chemically synthesized”, as related to a nucleic acidfragment, means that the component nucleotides were assembled in vitro.Manual chemical synthesis of nucleic acid fragments may be accomplishedusing well established procedures, or automated chemical synthesis canbe performed using one of a number of commercially available machines.Accordingly, the nucleic acid fragments can be tailored for optimal geneexpression based on optimization of the nucleotide sequence to reflectthe codon bias of the host cell. The skilled artisan appreciates thelikelihood of successful gene expression if codon usage is biasedtowards those codons favored by the host. Determination of preferredcodons can be based on a survey of genes derived from the host cellwhere sequence information is available.

[0037] “Gene” refers to a nucleic acid fragment that expresses aspecific protein, including regulatory sequences preceding (5′non-coding sequences) and following (3′ non-coding sequences) the codingsequence. “Native gene” refers to a gene as found in nature with its ownregulatory sequences. “Chimeric gene” refers any gene that is not anative gene, comprising regulatory and coding sequences that are notfound together in nature. Accordingly, a chimeric gene may compriseregulatory sequences and coding sequences that are derived fromdifferent sources, or regulatory sequences and coding sequences derivedfrom the same source, but arranged in a manner different than that foundin nature. “Endogenous gene” refers to a native gene in its naturallocation in the genome of an organism. A “foreign gene” refers to a genenot normally found in the host organism, but that is introduced into thehost organism by gene transfer. Foreign genes can comprise native genesinserted into a non-native organism, or chimeric genes. A “transgene” isa gene that has been introduced into the genome by a transformationprocedure.

[0038] “Coding sequence” refers to a nucleotide sequence that codes fora specific amino acid sequence. “Regulatory sequences” refers tonucleotide sequences located upstream (5′ non-coding sequences), within,or downstream (3′ non-coding sequences) of a coding sequence, and whichinfluence the transcription, RNA processing or stability, or translationof the associated coding sequence. Regulatory sequences may includepromoters, translation leader sequences, introns, and polyadenylationrecognition sequences.

[0039] “Promoter” refers to a nucleotide sequence capable of controllingthe expression of a coding sequence or functional RNA. In general, acoding sequence is located 3′ to a promoter sequence. The promotersequence consists of proximal and more distal upstream elements, thelatter elements often referred to as enhancers. Accordingly, an“enhancer” is a nucleotide sequence which can stimulate promoteractivity and may be an innate element of the promoter or a heterologouselement inserted to enhance the level or tissue-specificity of apromoter. Promoters may be derived in their entirety from a native gene,or may be composed of different elements derived from differentpromoters found in nature, or may even comprise synthetic nucleotidesegments. It is understood by those skilled in the art that differentpromoters may direct the expression of a gene in different tissues orcell types, or at different stages of development, or in response todifferent environmental conditions. Promoters which cause a nucleic acidfragment to be expressed in most cell types at most times are commonlyreferred to as “constitutive promoters”. New promoters of various typesuseful in plant cells are constantly being discovered; numerous examplesmay be found in the compilation by Okamuro and Goldberg (1989)Biochemistry of Plants 15:1-82. It is further recognized that since inmost cases the exact boundaries of regulatory sequences have not beencompletely defined, nucleic acid fragments of different lengths may haveidentical promoter activity.

[0040] “Translation leader sequence” refers to a nucleotide sequencelocated between the promoter sequence of a gene and the coding sequence.The translation leader sequence is present in the fully processed mRNAupstream of the translation start sequence. The translation leadersequence may affect processing of the primary transcript to mRNA, mRNAstability or translation efficiency. Examples of translation leadersequences have been described (Turner and Foster (1995) Mol. Biotechnol.3:225-236).

[0041] “3′ Non-coding sequences” refers to nucleotide sequences locateddownstream of a coding sequence and includes polyadenylation recognitionsequences and other sequences encoding regulatory signals capable ofaffecting mRNA processing or gene expression. The polyadenylation signalis usually characterized by affecting the addition of polyadenylic acidtracts to the 3′ end of the mRNA precursor. The use of different 3′non-coding sequences is exemplified by Ingelbrecht et al. (1989) PlantCell 1:671-680.

[0042] “RNA transcript” refers to the product resulting from RNApolymerase-catalyzed transcription of a DNA sequence. When the RNAtranscript is a perfect complementary copy of the DNA sequence, it isreferred to as the primary transcript or it may be a RNA sequencederived from posttranscriptional processing of the primary transcriptand is referred to as the mature RNA. “Messenger RNA (mRNA)” refers tothe RNA that is without introns and can be translated into polypeptidesby the cell. “cDNA” refers to DNA that is complementary to and derivedfrom an mRNA template. The cDNA can be single-stranded or converted todouble stranded form using, for example, the Klenow fragment of DNApolymerase I. “Sense RNA” refers to an RNA transcript that includes themRNA and can be translated into a polypeptide by the cell. “AntisenseRNA” refers to an RNA transcript that is complementary to all or part ofa target primary transcript or mRNA and that blocks the expression of atarget gene (see U.S. Pat. No. 5,107,065, incorporated herein byreference). The complementarity of an antisense RNA may be with any partof the specific nucleotide sequence, i.e., at the 5′ non-codingsequence, 3′ non-coding sequence, introns, or the coding sequence.“Functional RNA” refers to sense RNA, antisense RNA, ribozyme RNA, orother RNA that may not be translated but yet has an effect on cellularprocesses.

[0043] The term “operably linked” refers to the association of two ormore nucleic acid fragments so that the function of one is affected bythe other. For example, a promoter is operably linked with a codingsequence when it is capable of affecting the expression of that codingsequence (i.e., that the coding sequence is under the transcriptionalcontrol of the promoter). Coding sequences can be operably linked toregulatory sequences in sense or antisense orientation.

[0044] The term “expression”, as used herein, refers to thetranscription and stable accumulation of sense (mRNA) or antisense RNAderived from the nucleic acid fragment of the invention. “Expression”may also refer to translation of mRNA into a polypeptide. “Antisenseinhibition” refers to the production of antisense RNA transcriptscapable of suppressing the expression of the target protein.“Overexpression” refers to the production of a gene product intransgenic organisms that exceeds levels of production in normal ornon-transformed organisms. “Co-suppression” refers to the production ofsense RNA transcripts capable of suppressing the expression of identicalor substantially similar foreign or endogenous genes (U.S. Pat. No.5,231,020, incorporated herein by reference).

[0045] A “protein” or “polypeptide” is a chain of amino acids arrangedin a specific order determined by the coding sequence in apolynucleotide encoding the polypeptide. Each protein or polypeptide hasa unique function.

[0046] “Altered levels” or “altered expression” refer to the productionof gene product(s) in transgenic organisms in amounts or proportionsthat differ from that of normal or non-transformed organisms.

[0047] “Null mutant” refers to a host cell which either lacks theexpression of a certain polypeptide or expresses a polypeptide which isinactive or does not have any detectable expected enzymatic function.

[0048] “Mature protein” or the term “mature” when used in describing aprotein refers to a post-translationally processed polypeptide; i.e.,one from which any pre- or propeptides present in the primarytranslation product have been removed. “Precursor protein” or the term“precursor” when used in describing a protein refers to the primaryproduct of translation of mRNA; i.e., with pre- and propeptides stillpresent. Pre- and propeptides may be but are not limited tointracellular localization signals.

[0049] A “chloroplast transit peptide” is an amino acid sequence whichis translated in conjunction with a protein and directs the protein tothe chloroplast or other plastid types present in the cell in which theprotein is made. “Chloroplast transit sequence” refers to a nucleotidesequence that encodes a chloroplast transit peptide. A “signal peptide”is an amino acid sequence which is translated in conjunction with aprotein and directs the protein to the secretory system (Chrispeels(1991) Ann. Rev. Plant Phys. Plant Mol. Biol. 42:21-53). If the proteinis to be directed to a vacuole, a vacuolar targeting signal (supra) canfurther be added, or if to the endoplasmic reticulum, an endoplasmicreticulum retention signal (supra) may be added. If the protein is to bedirected to the nucleus, any signal peptide present should be removedand instead a nuclear localization signal included (Raikhel (1992) PlantPhys. 100:1627-1632).

[0050] “Transformation” refers to the transfer of a nucleic acidfragment into the genome of a host organism, resulting in geneticallystable inheritance. Host organisms containing the transformed nucleicacid fragments are referred to as “transgenic” organisms. Examples ofmethods of plant transformation include Agrobacterium-mediatedtransformation (De Blaere et al. (1987) Meth. Enzymol. 143:277) andparticle-accelerated or “gene gun” transformation technology (Klein etal. (1987) Nature (London) 327:70-73; U.S. Pat. No. 4,945,050,incorporated herein by reference). Thus, isolated polynucleotides of thepresent invention can be incorporated into recombinant constructs,typically DNA constructs, capable of introduction into and replicationin a host cell. Such a construct can be a vector that includes areplication system and sequences that are capable of transcription andtranslation of a polypeptide-encoding sequence in a given host cell. Anumber of vectors suitable for stable transfection of plant cells or forthe establishment of transgenic plants have been described in, e.g.,Pouwels et al., Cloning Vectors: A Laboratory Manual, 1985, supp. 1987;Weissbach and Weissbach, Methods for Plant Molecular Biology, AcademicPress, 1989; and Flevin et al., Plant Molecular Biology Manual, KluwerAcademic Publishers, 1990. Typically, plant expression vectors include,for example, one or more cloned plant genes under the transcriptionalcontrol of 5′ and 3′ regulatory sequences and a dominant selectablemarker. Such plant expression vectors also can contain a promoterregulatory region (e.g., a regulatory region controlling inducible orconstitutive, environmentally- or developmentally-regulated, or cell- ortissue-specific expression), a transcription initiation start site, aribosome binding site, an RNA processing signal, a transcriptiontermination site, and/or a polyadenylation signal.

[0051] Standard recombinant DNA and molecular cloning techniques usedherein are well known in the art and are described more fully inSambrook et al. Molecular Cloning: A Laboratory Manual; Cold SpringHarbor Laboratory Press: Cold Spring Harbor, 1989 (hereinafter“Maniatis”).

[0052] “PCR” or “polymerase chain reaction” is well known by thoseskilled in the art as a technique used for the amplification of specificDNA segments (U.S. Pat. Nos. 4,683,195 and 4,800,159).

[0053] The present invention concerns an isolated polynucleotidecomprising: (a) a nucleotide sequence encoding a polypeptide comprisingat least 133 amino acids, wherein the amino acid sequence of thepolypeptide and the amino acid sequence of SEQ ID NO: 2, 4, 6, 8, 10,12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46,or 48 have at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%identity based on the Clustal alignment method, (b) the complement ofthe nucleotide sequence, wherein the complement contains the same numberof nucleotides and is 100% complementary. The polypeptide preferablycomprises the amino acid sequence of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14,16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, or 48.The nucleotide sequence preferably comprises the nucleotide sequence ofSEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31,33, 35, 37, 39, 41, 43, 45, or 47. The polypeptide preferably is aribonuclease D (RNaseD).

[0054] The present invention concerns an isolated polynucleotidecomprising a nucleotide sequence selected from the group consisting of:(a) first nucleotide sequence encoding a polypeptide of at least 133amino acids having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,or 95% identity based on the Clustal method of alignment when comparedto a polypeptide selected from the group consisting of SEQ ID NO: 2, 4,6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40,42, 44, 46, or 48, and (b) a second nucleotide sequence comprising acomplement of the first nucleotide sequence.

[0055] Preferably, the nucleotide sequence comprises a nucleic acidsequence selected from the group consisting of SEQ ID NO: 1, 3, 5, 7, 9,11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45,or 47, that codes for the polypeptide selected from the group consistingof SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30,32, 34, 36, 38, 40, 42, 44, 46, or 48.

[0056] Nucleic acid fragments encoding at least a substantial portion ofseveral RNaseD-like proteins have been isolated and identified bycomparison of random plant cDNA sequences to public databases containingnucleotide and protein sequences using the BLAST algorithms well knownto those skilled in the art. The nucleic acid fragments of the instantinvention may be used to isolate cDNAs and genes encoding homologousproteins from the same or other plant species. Isolation of homologousgenes using sequence-dependent protocols is well known in the art.Examples of sequence-dependent protocols include, but are not limitedto, methods of nucleic acid hybridization, and methods of DNA and RNAamplification as exemplified by various uses of nucleic acidamplification technologies (e.g., polymerase chain reaction, ligasechain reaction).

[0057] For example, genes encoding other RNaseD-like proteins, either ascDNAs or genomic DNAs, could be isolated directly by using all or asubstantial portion of the instant nucleic acid fragments as DNAhybridization probes to screen libraries from any desired plantemploying methodology well known to those skilled in the art. Specificoligonucleotide probes based upon the instant nucleic acid sequences canbe designed and synthesized by methods known in the art (Maniatis).Moreover, an entire sequence(s) can be used directly to synthesize DNAprobes by methods known to the skilled artisan such as random primer DNAlabeling, nick translation, end-labeling techniques, or RNA probes usingavailable in vitro transcription systems. In addition, specific primerscan be designed and used to amplify a part or all of the instantsequences. The resulting amplification products can be labeled directlyduring amplification reactions or labeled after amplification reactions,and used as probes to isolate full length cDNA or genomic fragmentsunder conditions of appropriate stringency.

[0058] In addition, two short segments of the instant nucleic acidfragments may be used in polymerase chain reaction protocols to amplifylonger nucleic acid fragments encoding homologous genes from DNA or RNA.The polymerase chain reaction may also be performed on a library ofcloned nucleic acid fragments wherein the sequence of one primer isderived from the instant nucleic acid fragments, and the sequence of theother primer takes advantage of the presence of the polyadenylic acidtracts to the 3′ end of the mRNA precursor encoding plant genes.Alternatively, the second primer sequence may be based upon sequencesderived from the cloning vector. For example, the skilled artisan canfollow the RACE protocol (Frohman et al. (1988) Proc. Natl. Acad. Sci.USA 85:8998-9002) to generate cDNAs by using PCR to amplify copies ofthe region between a single point in the transcript and the 3′ or 5′end. Primers oriented in the 3′ and 5′ directions can be designed fromthe instant sequences. Using commercially available 3′ RACE or 5′ RACEsystems (BRL), specific 3′ or 5′ cDNA fragments can be isolated (Oharaet al. (1989) Proc. Natl. Acad. Sci. USA 86:5673-5677; Loh et al. (1989)Science 243:217-220). Products generated by the 3′ and 5′ RACEprocedures can be combined to generate full-length cDNAs (Frohman andMartin (1989) Techniques 1:165). Consequently, a polynucleotidecomprising a nucleotide sequence of at least one of 30 (preferably oneof at least 40, most preferably one of at least 60) contiguousnucleotides derived from a nucleotide sequence selected from the groupconsisting of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25,27, 29, 31, 33, 35, 37, 39, 41, 43, 45, or 47, and the complement ofsuch nucleotide sequences may be used in such methods to obtain anucleic acid fragment encoding a substantial portion of an amino acidsequence of a polypeptide.

[0059] The present invention relates to a method of obtaining a nucleicacid fragment encoding a substantial portion of a RNaseD-likepolypeptide, preferably a substantial portion of a plant RNaseD-likepolypeptide, comprising the steps of: synthesizing an oligonucleotideprimer comprising a nucleotide sequence of at least one of 30(preferably at least one of 40, most preferably at least one of 60)contiguous nucleotides derived from a nucleotide sequence selected fromthe group consisting of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19,21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45,or 47,and thecomplement of such nucleotide sequences; and amplifying a nucleic acidfragment (preferably a cDNA inserted in a cloning vector) using theoligonucleotide primer. The amplified nucleic acid fragment preferablywill encode a substantial portion of a RNaseD-like polypeptide.

[0060] Availability of the instant nucleotide and deduced amino acidsequences facilitates immunological screening of cDNA expressionlibraries. Synthetic peptides representing substantial portions of theinstant amino acid sequences may be synthesized. These peptides can beused to immunize animals to produce polyclonal or monoclonal antibodieswith specificity for peptides or proteins comprising the amino acidsequences. These antibodies can be then be used to screen cDNAexpression libraries to isolate full-length cDNA clones of interest(Lerner (1984) Adv. Immunol. 36:1-34; Maniatis).

[0061] In another embodiment, this invention concerns viruses and hostcells comprising either the chimeric genes of the invention as describedherein or an isolated polynucleotide of the invention as describedherein. Examples of host cells which can be used to practice theinvention include, but are not limited to, yeast, bacteria, and plants.

[0062] As was noted above, the nucleic acid fragments of the instantinvention may be used to create transgenic plants in which the disclosedpolypeptides are present at higher or lower levels than normal or incell types or developmental stages in which they are not normally found.This would have the effect of altering the level of gene expression inthose cells.

[0063] Overexpression of the proteins of the instant invention may beaccomplished by first constructing a chimeric gene in which the codingregion is operably linked to a promoter capable of directing expressionof a gene in the desired tissues at the desired stage of development.The chimeric gene may comprise promoter sequences and translation leadersequences derived from the same genes. 3′ Non-coding sequences encodingtranscription termination signals may also be provided. The instantchimeric gene may also comprise one or more introns in order tofacilitate gene expression.

[0064] Plasmid vectors comprising the instant isolated polynucleotide(or chimeric gene) may be constructed. The choice of plasmid vector isdependent upon the method that will be used to transform host plants.The skilled artisan is well aware of the genetic elements that must bepresent on the plasmid vector in order to successfully transform, selectand propagate host cells containing the chimeric gene. The skilledartisan will also recognize that different independent transformationevents will result in different levels and patterns of expression (Joneset al. (1985) EMBO J. 4:2411-2418; De Almeida et al. (1989) Mol. Gen.Genetics 218:78-86), and thus that multiple events must be screened inorder to obtain lines displaying the desired expression level andpattern. Such screening may be accomplished by Southern analysis of DNA,Northern analysis of mRNA expression, Western analysis of proteinexpression, or phenotypic analysis.

[0065] For some applications it may be useful to direct the instantpolypeptides to different cellular compartments, or to facilitate theirsecretion from the cell. It is thus envisioned that the chimeric genedescribed above may be further supplemented by directing the codingsequence to encode the instant polypeptides with appropriateintracellular targeting sequences such as transit sequences (Keegstra(1989) Cell 56:247-253), signal sequences or sequences encodingendoplasmic reticulum localization (Chrispeels (1991) Ann. Rev. PlantPhys. Plant Mol. Biol. 42:21-53), or nuclear localization signals(Raikhel (1992) Plant Phys. 100:1627-1632) with or without removingtargeting sequences that are already present. While the references citedgive examples of each of these, the list is not exhaustive and moretargeting signals of use may be discovered in the future.

[0066] It may also be desirable to reduce or eliminate expression ofgenes encoding the instant polypeptides in plants for some applications.In order to accomplish this, a chimeric gene designed for co-suppressionof the instant polypeptide can be constructed by linking a gene or genefragment encoding that polypeptide to plant promoter sequences.Alternatively, a chimeric gene designed to express antisense RNA for allor part of the instant nucleic acid fragment can be constructed bylinking the gene or gene fragment in reverse orientation to plantpromoter sequences. Either the co-suppression or antisense chimericgenes could be introduced into plants via transformation whereinexpression of the corresponding endogenous genes are reduced oreliminated.

[0067] Molecular genetic solutions to the generation of plants withaltered gene expression have a decided advantage over more traditionalplant breeding approaches. Changes in plant phenotypes can be producedby specifically inhibiting expression of one or more genes by antisenseinhibition or cosuppression (U.S. Pat. Nos. 5,190,931, 5,107,065 and5,283,323). An antisense or cosuppression construct would act as adominant negative regulator of gene activity. While conventionalmutations can yield negative regulation of gene activity these effectsare most likely recessive. The dominant negative regulation availablewith a transgenic approach may be advantageous from a breedingperspective. In addition, the ability to restrict the expression of aspecific phenotype to the reproductive tissues of the plant by the useof tissue specific promoters may confer agronomic advantages relative toconventional mutations which may have an effect in all tissues in whicha mutant gene is ordinarily expressed.

[0068] The person skilled in the art will know that specialconsiderations are associated with the use of antisense or cosuppressiontechnologies in order to reduce expression of particular genes. Forexample, the proper level of expression of sense or antisense genes mayrequire the use of different chimeric genes utilizing differentregulatory elements known to the skilled artisan. Once transgenic plantsare obtained by one of the methods described above, it will be necessaryto screen individual transgenics for those that most effectively displaythe desired phenotype. Accordingly, the skilled artisan will developmethods for screening large numbers of transformants. The nature ofthese screens will generally be chosen on practical grounds. Forexample, one can screen by looking for changes in gene expression byusing antibodies specific for the protein encoded by the gene beingsuppressed, or one could establish assays that specifically measureenzyme activity. A preferred method will be one which allows largenumbers of samples to be processed rapidly, since it will be expectedthat a large number of transformants will be negative for the desiredphenotype.

[0069] RNaseD enzymes have been implicated in the production of shortRNA species (22-25 nucleotides) that may be double-stranded in nature(Fagard and Vaucheret (2000) Plant Mol Biol 43:295-306). In addition toplaying a role in gene silencing or cosuppression, the generation ofthese short RNA species is involved in plant defenses against viruses(Baulcombe (1996) Plant Mol Biol 32:79-88). Specifically, production ofthese short viral RNA species in a plant results in resistance of theplant to viral infection. Therefore, over-expression in plants of theRNaseD enzymes of the present invention will increase the resistance ofthese plants to viral infection.

[0070] The present invention concerns an isolated polypeptide comprisingan amino acid sequence comprising at least 133 amino acids, wherein theamino acid sequence and the amino acid sequence of SEQ ID NO:2, 4, 6, 8,10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44,46, or 48 have at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or95 % identity based on the Clustal alignment method. The first aminoacid sequence preferably comprises the amino acid sequence of SEQ IDNO:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36,38, 40, 42, 44, 46, or 48. The polypeptide preferably has RnaseDactivity.

[0071] The instant polypeptides (or substantial portions thereof) may beproduced in heterologous host cells, particularly in the cells ofmicrobial hosts, and can be used to prepare antibodies these proteins bymethods well known to those skilled in the art. The antibodies areuseful for detecting the polypeptides of the instant invention in situin cells or in vitro in cell extracts. Preferred heterologous host cellsfor production of the instant polypeptides are microbial hosts.Microbial expression systems and expression vectors containingregulatory sequences that direct high level expression of foreignproteins are well known to those skilled in the art. Any of these couldbe used to construct a chimeric gene for production of the instantpolypeptides. This chimeric gene could then be introduced intoappropriate microorganisms via transformation to provide high levelexpression of the encoded RNaseD-like. An example of a vector for highlevel expression of the instant polypeptides in a bacterial host isprovided (Example 6).

[0072] All or a substantial portion of the polynucleotides of theinstant invention may also be used as probes for genetically andphysically mapping the genes that they are a part of, and used asmarkers for traits linked to those genes. Such information may be usefulin plant breeding in order to develop lines with desired phenotypes. Forexample, the instant nucleic acid fragments may be used as restrictionfragment length polymorphism (RFLP) markers. Southern blots (Maniatis)of restriction-digested plant genomic DNA may be probed with the nucleicacid fragments of the instant invention. The resulting banding patternsmay then be subjected to genetic analyses using computer programs suchas MapMaker (Lander et al. (1987) Genomics 1:174-181) in order toconstruct a genetic map. In addition, the nucleic acid fragments of theinstant invention may be used to probe Southern blots containingrestriction endonuclease-treated genomic DNAs of a set of individualsrepresenting parent and progeny of a defined genetic cross. Segregationof the DNA polymorphisms is noted and used to calculate the position ofthe instant nucleic acid sequence in the genetic map previously obtainedusing this population (Botstein et al. (1980) Am. J. Hum. Genet.32:314-331).

[0073] The production and use of plant gene-derived probes for use ingenetic mapping is described in Bernatzky and Tanksley (1986) Plant Mol.Biol. Reporter 4:37-41. Numerous publications describe genetic mappingof specific cDNA clones using the methodology outlined above orvariations thereof. For example, F2 intercross populations, backcrosspopulations, randomly mated populations, near isogenic lines, and othersets of individuals may be used for mapping. Such methodologies are wellknown to those skilled in the art.

[0074] Nucleic acid probes derived from the instant nucleic acidsequences may also be used for physical mapping (i.e., placement ofsequences on physical maps; see Hoheisel et al. In: Nonmammalian GenomicAnalysis: A Practical Guide, Academic press 1996, pp. 319-346, andreferences cited therein).

[0075] In another embodiment, nucleic acid probes derived from theinstant nucleic acid sequences may be used in direct fluorescence insitu hybridization (FISH) mapping (Trask 30 (1991) Trends Genet.7:149-154). Although current methods of FISH mapping favor use of largeclones (several to several hundred KB; see Laan et al. (1995) GenomeRes. 5:13-20), improvements in sensitivity may allow performance of FISHmapping using shorter probes.

[0076] A variety of nucleic acid amplification-based methods of geneticand physical mapping may be carried out using the instant nucleic acidsequences. Examples include allele-specific amplification (Kazazian(1989) J. Lab. Clin. Med. 11:95-96), polymorphism of PCR-amplifiedfragments (CAPS; Sheffield et al. (1993) Genomics 16:325-332),allele-specific ligation (Landegren et al. (1988) Science241:1077-1080), nucleotide extension reactions (Sokolov (1990) NucleicAcid Res. 18:3671), Radiation Hybrid Mapping (Walter et al. (1997) Nat.Genet. 7:22-28) and Happy Mapping (Dear and Cook (1989) Nucleic AcidRes. 1 7:6795-6807). For these methods, the sequence of a nucleic acidfragment is used to design and produce primer pairs for use in theamplification reaction or in primer extension reactions. The design ofsuch primers is well known to those skilled in the art. In methodsemploying PCR-based genetic mapping, it may be necessary to identify DNAsequence differences between the parents of the mapping cross in theregion corresponding to the instant nucleic acid sequence. This,however, is generally not necessary for mapping methods.

[0077] Loss of function mutant phenotypes may be identified for theinstant cDNA clones either by targeted gene disruption protocols or byidentifying specific mutants for these genes contained in a maizepopulation carrying mutations in all possible genes (Ballinger andBenzer (1989) Proc. Natl. Acad. Sci USA 86:9402-9406; Koes et al. (1995)Proc. Natl. Acad. Sci USA 92:8149-8153; Bensen et al. (1995) Plant Cell7:75-84). The latter approach may be accomplished in two ways. First,short segments of the instant nucleic acid fragments may be used inpolymerase chain reaction protocols in conjunction with a mutation tagsequence primer on DNAs prepared from a population of plants in whichMutator transposons or some other mutation-causing DNA element has beenintroduced (see Bensen, supra). The amplification of a specific DNAfragment with these primers indicates the insertion of the mutation tagelement in or near the plant gene encoding the instant polypeptides.Alternatively, the instant nucleic acid fragment may be used as ahybridization probe against PCR amplification products generated fromthe mutation population using the mutation tag sequence primer inconjunction with an arbitrary genomic site primer, such as that for arestriction enzyme site-anchored synthetic adaptor. With either method,a plant containing a mutation in the endogenous gene encoding theinstant polypeptides can be identified and obtained. This mutant plantcan then be used to determine or confirm the natural function of theinstant polypeptides disclosed herein.

EXAMPLES

[0078] The present invention is further defined in the followingExamples, in which parts and percentages are by weight and degrees areCelsius, unless otherwise stated. It should be understood that theseExamples, while indicating preferred embodiments of the invention, aregiven by way of illustration only. From the above discussion and theseExamples, one skilled in the art can ascertain the essentialcharacteristics of this invention, and without departing from the spiritand scope thereof, can make various changes and modifications of theinvention to adapt it to various usages and conditions. Thus, variousmodifications of the invention in addition to those shown and describedherein will be apparent to those skilled in the art from the foregoingdescription. Such modifications are also intended to fall within thescope of the appended claims.

[0079] The disclosure of each reference set forth herein is incorporatedherein by reference in its entirety.

EXAMPLE 1 Composition of cDNA Libraries, Isolation and Sequencing ofcDNA Clones

[0080] cDNA libraries representing mRNAs from various corn, rice,soybean, Vernonia and wheat tissues were prepared. The characteristicsof the libraries are described below. TABLE 2 cDNA Libraries from Corn,Rice, Soybean, Vernonia and Wheat Library Tissue Clone cco1n Corn cob of67 day old plants grown in cco1n.pk058.p8 green house* cco1n.pk063.p21cco1n.pk094.o1 p0010 Corn log phase suspension cells treatedp0010.cbpbx36r with A23187 ® to induce mass apoptosis** p0018 Cornseedling after 10 day drought, heat p0018.chstb24r shocked for 24 hours,harvested after recovery at normal growth conditions for 8 hours p0046Corn shoots two and three days after p0046.cndai04r germination. p0098Corn ear shoot, prophasei (2.8-4.8 cm)* p0098.cdfao60r p0133 Corn pooledmeristem tissue @ V4, p0133.ctvad13r V6 and V8*** rca1n Rice callus*rca1n.pk023.m3 scr1c Soybean embryogenic suspension culturescr1c.pk004.m6 subjected to 4 vacuum cycles and collected 12 hours latersrc3c Soybean 8 day old root infected with cyst src3c.pk013.c12 nematodeHeterodera glycenes vs1 Vernonia seed vs1.pk0011.b11 w1mk8 Wheatseedlings 8 hours after inoculation w1mk8.pk0022.f7 with Erysiphegraminis f. sp tritici and treatment with herbicide**** wne1g Wheatnebulized genomic library wne1g.pk005.e5 cepe7 Corn 7 Day Old EpicotylFrom Etiolated cepe7.pk0008.b7:fis Seedling p0128 Pooled primary andsecondary immature p0128.cpicn88r ear eav1c Columbine (Aquilegiavulgaris) develop- eav1c.pk005.f18:fis ing seeds (looking for delta 5desaturase genes) vmb1c Grape (Vitis sp.) midstage berriesvmb1c.pk010.17:fis csi1n Corn Silk* csi1n.pk0017.f10:fis ehb2c Pararubber tree (Hevea brasiliensis, ehb2c.pk006.p12:fis PR255) latex tappedin 2nd day of 3 day tapping cycle rdi2c Rice (Oryza sativa, Nipponbare)develop- rdi2c.pk011.f3:fis ing inflorescence at rachis branch-floralorgan primordia formation scn1c Soybean (Glycine max L., 6705)scn1c.pk001.p19:fis Embryogenic suspension culture collected 10 monthsold (necrotic tissue). sea1c Soybean (Glycine max, A2396) embryonicsea1c.pk015.i18 axis dissected from seeds imbibed overnight sgs1cSoybean Seeds 4 Hours After Germination sgs1c.pk003.h10 hss1cSclerotinia infected sunflower plants, hss1c.pk015.a23 purpose isolationof full length Sclerotinia induced cDNAs

[0081] cDNA libraries may be prepared by any one of many methodsavailable. For example, the cDNAs may be introduced into plasmid vectorsby first preparing the cDNA libraries in Uni-ZAP™ XR vectors accordingto the manufacturer's protocol (Stratagene Cloning Systems, La Jolla,Calif.). The Uni-ZAP™ XR libraries are converted into plasmid librariesaccording to the protocol provided by Stratagene. Upon conversion, cDNAinserts will be contained in the plasmid vector pBluescript. Inaddition, the cDNAs may be introduced directly into precut Bluescript IISK(+) vectors (Stratagene) using T4 DNA ligase (New England Biolabs),followed by transfection into DH10B cells according to themanufacturer's protocol (GIBCO BRL Products). Once the cDNA inserts arein plasmid vectors, plasmid DNAs are prepared from randomly pickedbacterial colonies containing recombinant pBluescript plasmids, or theinsert cDNA sequences are amplified via polymerase chain reaction usingprimers specific for vector sequences flanking the inserted cDNAsequences. Amplified insert DNAs or plasmid DNAs are sequenced indye-primer sequencing reactions to generate partial cDNA sequences(expressed sequence tags or “ESTs”; see Adams et al., (1991) Science252:1651-1656). The resulting ESTs are analyzed using a Perkin ElmerModel 377 fluorescent sequencer.

EXAMPLE 2 Identification of cDNA Clones

[0082] cDNA clones encoding RNaseD-like proteins were identified byconducting BLAST (Basic Local Alignment Search Tool; Altschul et al.(1993) J. Mol. Biol. 215:403-410; see also www.ncbi.nlm.nih.gov/BLAST/)searches for similarity to sequences contained in the BLAST “nr”database (comprising all non-redundant GenBank CDS translations,sequences derived from the 3-dimensional structure Brookhaven ProteinData Bank, the last major release of the SWISS-PROT protein sequencedatabase, EMBL, and DDBJ databases). The cDNA sequences obtained inExample 1 were analyzed for similarity to all publicly available DNAsequences contained in the “nr” database using the BLASTN algorithmprovided by the National Center for Biotechnology Information (NCBI).The DNA sequences were translated in all reading frames and compared forsimilarity to all publicly available protein sequences contained in the“nr” database using the BLASTX algorithm (Gish and States (1993) Nat.Genet. 3:266-272) provided by the NCBI. For convenience, the P-value(probability) of observing a match of a cDNA sequence to a sequencecontained in the searched databases merely by chance as calculated byBLAST are reported herein as “pLog” values, which represent the negativeof the logarithm of the reported P-value. Accordingly, the greater thepLog value, the greater the likelihood that the cDNA sequence and theBLAST “hit” represent homologous proteins.

EXAMPLE 3 Characterization of cDNA Clones Encoding RNaseD-like Proteins

[0083] The BLASTX search using the EST sequences from clones listed inTable 3 revealed similarity of the polypeptides encoded by the cDNAs toRNaseD-like proteins from Arabidopsis thaliana (NCBI General IdentifierNo. gi 4455316), Arabidopsis thaliana (NCBI General Identifier No. gi7845693), Arabidopsis thaliana (NCBI General Identifier No. gi 3298537,4585987, 12321757, 12321965, 10177895, and 10177896), Mus musculus (NCBIGeneral Identifier No. gi 2645409), Pyrococcus horikoshii (NCB GeneralIdentifier No. gi 7518800), Pyrococcus abyssi (NCB General IdentifierNo. gi 7518144) and Caenorhabditis elegans (NCB General Identifier No.gi 466063). Shown in Table 3 are the BLAST results for individual ESTs(“EST”), the sequences of the entire cDNA inserts comprising theindicated cDNA clones (“FIS”), the sequences of contigs assembled fromtwo or more ESTs (“Contig”), sequences of contigs assembled from an FISand one or more ESTs (“Contig*”), or sequences encoding an entireprotein derived from an FIS, a contig, or an FIS and PCR (“CGS”): TABLE3 BLAST Results for Sequences Encoding Polypeptides Homologous toArabidopsis thaliana, Mus musculus, Pyrococcus horikoshii, Pyrococcusabyssi and Caenorhabditis elegans RNaseD-like Proteins BLAST pLog ScoreClone Status (NCBI General Identifier No.) cco1n.pk063.p21 FIS 17.00 (gi2645409) Contig composed of ESTs: Contig 15.52 (gi 4455316)cco1n.pk058.p8 cco1n.pk094.o1 p0010.cbpbx36r p0098.cdfao60rp0018.chstb24r FIS 9.15 (gi 466063) p0046.cndai04r FIS 10.00 (gi7518800) p0133.ctvad13r FIS 18.70 (gi 7485693) rca1n.pk023.m3 FIS 18.15(gi 7485693) scr1c.pk004.m6 FIS 20.05 (gi 7485693) src3c.pk013.c12 FIS64.15 (gi 7485693) vs1.pk0011.b11 FIS 10.30 (gi 7518144) w1mk8.pk0022.f7FIS 6.70 (gi 7518800) wne1g.pk005.e5 EST 4.30 (gi 3298537)cco1n.pk063.p21:fis FIS 17.0 (gi 2645409) cepe7.pk0008.b7:fis FIS 133.0(gi 12321757) contig of: Contig 90.0 (gi 10177896) p0046.cndai04r:fis,p0128.cpicn88r w1mk8.pk0022.f7:fis FIS 129.0 (gi 10177895)eav1c.pk005.f18:fis FIS 12.0 (gi 7485693) vmb1c.pk010.17:fis FIS 106.0(gi 4585987) csi1n.pk0017.f10:fis FIS 13.3 (gi 12321965)ehb2c.pk006.p12:fis FIS 21.7 (gi 7485693) rdi2c.pk011.f3:fis FIS 22.5(gi 12321965) scn1c.pk001.p19:fis FIS 176.0 (gi 12321757)sea1c.pk015.i18 EST 36.5 (gi 10177895) sgs1c.pk003.h10 EST 25.3 (gi10177895) hss1c.pk015.a23 EST 54.5 (gi 10177895)

[0084] The data in Table 4 represents a calculation of the percentidentity of the amino acid sequences set forth in SEQ ID NOs: 2, 4, 6,8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42,44, 46, and 48 and the Arabidopsis thaliana, Mus musculus, Pyrococcushorikoshii, Pyrococcus abyssi and Caenorhabditis elegans sequences.TABLE 4 Percent Identity of Amino Acid Sequences Deduced From theNucleotide Sequences of cDNA Clones Encoding Polypeptides Homologous toArabidopsis thaliana, Mus musculus, Pyrococcus horikoshii, Pyrococcusabyssi and Caenorhabditis elegans RNaseD-like Proteins Percent Identityto SEQ ID NO. (NCBI General Identifier No.) 2 24% (gi 2645409) 4 21% (gi4455316) 6 29% (gi 466063) 8 23% (gi 7518800) 10 23% (gi 7485693) 12 24%(gi 7485693) 14 23% (gi 7485693) 16 42% (gi 7485693) 18 25% (gi 7518144)20 22% (gi 7518800) 22 19% (gi 3298537) 24 24% (gi 2645409) 26 41% (gi12321757) 28 45% (gi 10177895) 30 49.8% (gi 10177895) 32 47.7% (gi12321757) 34 13.5% (gi 12321965) 36 20% (gi 12321965) 38 24% (gi7485693) 40 27% (gi 12321965) 42 52% (gi 12321757) 44 88% (gi 10177895)46 44% (gi 10177895) 48 51.5% (gi 10177895)

[0085] Sequence alignments and percent identity calculations wereperformed using the Megalign program of the LASERGENE bioinformaticscomputing suite (DNASTAR Inc., Madison, Wis.) Multiple alignment of thesequences was performed using the Clustal method of alignment (Higginsand Sharp (1989) CABIOS. 5:151 - 153) with the default parmeters (GAPPENALTY=10, GAP LENGTH PENALTY=10). Default parameters for pairwisealiments using the Clustal method were KTUPLE 1, GAP PENALTY=3, WINDOW=5and DIAGONALS SAVED=5. Sequence alignments, BLAST scores andprobabilities indicate that the nucleic acid fragments comprising theinstant cDNA clones encode a substantial portion of a RNaseD-likeprotein.

EXAMPLE 4 Expression of Chimeric Genes in Monocot Cells

[0086] A chimeric gene comprising a cDNA encoding the instantpolypeptides in sense orientation with respect to the maize 27 kD zeinpromoter that is located 5′ to the cDNA fragment, and the 10 kD zein 3′end that is located 3′ to the cDNA fragment, can be constructed. ThecDNA fragment of this gene may be generated by polymerase chain reaction(PCR) of the cDNA clone using appropriate oligonucleotide primers.Cloning sites (NcoI or Smal) can be incorporated into theoligonucleotides to provide proper orientation of the DNA fragment wheninserted into the digested vector pML103 as described below.Amplification is then performed in a standard PCR. The amplified DNA isthen digested with restriction enzymes NcoI and Smal and fractionated onan agarose gel. The appropriate band can be isolated from the gel andcombined with a 4.9 kb NcoI-SmaI fragment of the plasmid pML103. PlasmidpML103 has been deposited under the terms of the Budapest Treaty at ATCC(American Type Culture Collection, 10801 University Blvd., Manassas, Va.20110-2209), and bears accession number ATCC 97366. The DNA segment frompML103 contains a 1.05 kb SalI-NcoI promoter fragment of the maize 27 kDzein gene and a 0.96 kb SmaI-SalI fragment from the 3′ end of the maize10 kD zein gene in the vector pGem9Zf(+) (Promega). Vector and insertDNA can be ligated at 15° C. overnight, essentially as described(Maniatis). The ligated DNA may then be used to transform E. coliXL1-Blue (Epicurian Coli XL-1 Blue™; Stratagene). Bacterialtransformants can be screened by restriction enzyme digestion of plasmidDNA and limited nucleotide sequence analysis using the dideoxy chaintermination method (Sequenase™ DNA Sequencing Kit; U.S. Biochemical).The resulting plasmid construct would comprise a chimeric gene encoding,in the 5′ to 3′ direction, the maize 27 kD zein promoter, a cDNAfragment encoding the instant polypeptides, and the 10 kD zein 3′region.

[0087] The chimeric gene described above can then be introduced intocorn cells by the following procedure. Immature corn embryos can bedissected from developing caryopses derived from crosses of the inbredcorn lines H99 and LH132. The embryos are isolated 10 to 11 days afterpollination when they are 1.0 to 1.5 mm long. The embryos are thenplaced with the axis-side facing down and in contact withagarose-solidified N6 medium (Chu et al. (1975) Sci. Sin. Peking18:659-668). The embryos are kept in the dark at 27° C. Friableembryogenic callus consisting of undifferentiated masses of cells withsomatic proembryoids and embryoids borne on suspensor structuresproliferates from the scutellum of these immature embryos. Theembryogenic callus isolated from the primary explant can be cultured onN6 medium and sub-cultured on this medium every 2 to 3 weeks.

[0088] The plasmid, p35S/Ac (obtained from Dr. Peter Eckes, Hoechst Ag,Frankfurt, Germany) may be used in transformation experiments in orderto provide for a selectable marker. This plasmid contains the Pat gene(see European Patent Publication 0 242 236) which encodesphosphinothricin acetyl transferase (PAT). The enzyme PAT confersresistance to herbicidal glutamine synthetase inhibitors such asphosphinothricin. The pat gene in p35S/Ac is under the control of the35S promoter from Cauliflower Mosaic Virus (Odell et al. (1985) Nature313:810-812) and the 3′ region of the nopaline synthase gene from theT-DNA of the Ti plasmid of Agrobacterium tumefaciens.

[0089] The particle bombardment method (Klein et al. (1987) Nature327:70-73) may be used to transfer genes to the callus culture cells.According to this method, gold particles (1 μm in diameter) are coatedwith DNA using the following technique. Ten μg of plasmid DNAs are addedto 50 μL of a suspension of gold particles (60 mg per mL). Calciumchloride (50 μL of a 2.5 M solution) and spermidine free base (20 μL ofa 1.0 M solution) are added to the particles. The suspension is vortexedduring the addition of these solutions. After 10 minutes, the tubes arebriefly centrifuged (5 sec at 15,000 rpm) and the supernatant removed.The particles are resuspended in 200 μL of absolute ethanol, centrifugedagain and the supernatant removed. The ethanol rinse is performed againand the particles resuspended in a final volume of 30 μL of ethanol. Analiquot (5 μL) of the DNA-coated gold particles can be placed in thecenter of a Kapton™ flying disc (Bio-Rad Labs). The particles are thenaccelerated into the corn tissue with a Biolistic™ PDS-1000/He (Bio-RadInstruments, Hercules Calif.), using a helium pressure of 1000 psi, agap distance of 0.5 cm and a flying distance of 1.0 cm.

[0090] For bombardment, the embryogenic tissue is placed on filter paperover agarose-solidified N6 medium. The tissue is arranged as a thin lawnand covered a circular area of about 5 cm in diameter. The petri dishcontaining the tissue can be placed in the chamber of the PDS-1000/Heapproximately 8 cm from the stopping screen. The air in the chamber isthen evacuated to a vacuum of 28 inches of mercury (Hg). Themacrocarrier is accelerated with a helium shock wave using a rupturemembrane that bursts when the He pressure in the shock tube reaches 1000psi.

[0091] Seven days after bombardment the tissue can be transferred to N6medium that contains gluphosinate (2 mg per liter) and lacks casein orproline. The tissue continues to grow slowly on this medium. After anadditional 2 weeks the tissue can be transferred to fresh N6 mediumcontaining gluphosinate. After 6 weeks, areas of about 1 cm in diameterof actively growing callus can be identified on some of the platescontaining the glufosinate-supplemented medium. These calli may continueto grow when sub-cultured on the selective medium.

[0092] Plants can be regenerated from the transgenic callus by firsttransferring clusters of tissue to N6 medium supplemented with 0.2 mgper liter of 2,4-D. After two weeks the tissue can be transferred toregeneration medium (Fromm et al. (1990) Bio/Technology 8:833-839).

EXAMPLE 5 Expression of Chimeric Genes in Dicot Cells

[0093] A seed-specific construct composed of the promoter andtranscription terminator from the gene encoding the β subunit of theseed storage protein phaseolin from the bean Phaseolus vulgaris (Doyleet al. (1986) J. Biol. Chem. 261:9228-9238) can be used for expressionof the instant polypeptides in transformed soybean. The phaseolinconstruct includes about 500 nucleotides upstream (5′) from thetranslation initiation codon and about 1650 nucleotides downstream (3′)from the translation stop codon of phaseolin. Between the 5′ and 3′regions are the unique restriction endonuclease sites Nco I (whichincludes the ATG translation initiation codon), Sma I, Kpn I and Xba I.The entire construct is flanked by Hind III sites.

[0094] The cDNA fragment of this gene may be generated by polymerasechain reaction (PCR) of the cDNA clone using appropriate oligonucleotideprimers. Cloning sites can be incorporated into the oligonucleotides toprovide proper orientation of the DNA fragment when inserted into theexpression vector. Amplification is then performed as described above,and the isolated fragment is inserted into a pUC18 vector carrying theseed construct.

[0095] Soybean embryos may then be transformed with the expressionvector comprising sequences encoding the instant polypeptides. To inducesomatic embryos, cotyledons, 3-5 mm in length dissected from surfacesterilized, immature seeds of the soybean cultivar A2872, can becultured in the light or dark at 26° C. on an appropriate agar mediumfor 6-10 weeks. Somatic embryos which produce secondary embryos are thenexcised and placed into a suitable liquid medium. After repeatedselection for clusters of somatic embryos which multiplied as early,globular staged embryos, the suspensions are maintained as describedbelow.

[0096] Soybean embryogenic suspension cultures can be maintained in 35mL of liquid media on a rotary shaker, 150 rpm, at 26° C. withflorescent lights on a 16:8 hour day/night schedule. Cultures aresubcultured every two weeks by inoculating approximately 35 mg of tissueinto 35 mL of liquid medium.

[0097] Soybean embryogenic suspension cultures may then be transformedby the method of particle gun bombardment (Klein et al. (1987) Nature(London) 327:70-73, U.S. Pat. No. 4,945,050). A DuPont Biolistic™ PDS1000/HE instrument (helium retrofit) can be used for thesetransformations.

[0098] A selectable marker gene which can be used to facilitate soybeantransformation is a chimeric gene composed of the 35S promoter fromCauliflower Mosaic Virus (Odell et al. (1985) Nature 313:810-812), thehygromycin phosphotransferase gene from plasmid pJR225 (from E. coli;Gritz et al.(1983) Gene 25:179-188) and the 3′ region of the nopalinesynthase gene from the T-DNA of the Ti plasmid of Agrobacteriumtumefaciens. The seed construct comprising the phaseolin 5′ region, thefragment encoding the instant polypeptides and the phaseolin 3′ regioncan be isolated as a restriction fragment. This fragment can then beinserted into a unique restriction site of the vector carrying themarker gene.

[0099] To 50 μL of a 60 mg/mL 1 μm gold particle suspension is added (inorder): 5 μL DNA (1 μg/μL), 20 μL spermidine (0.1 M), and 50 μL CaCl₂(2.5 M). The particle preparation is then agitated for three minutes,spun in a microfuge for 10 seconds and the supernatant removed. TheDNA-coated particles are then washed once in 400 μL 70% ethanol andresuspended in 40 μL of anhydrous ethanol. The DNA/particle suspensioncan be sonicated three times for one second each. Five μL of theDNA-coated gold particles are then loaded on each macro carrier disk.

[0100] Approximately 300-400 mg of a two-week-old suspension culture isplaced in an empty 60×15 mm petri dish and the residual liquid removedfrom the tissue with a pipette. For each transformation experiment,approximately 5-10 plates of tissue are normally bombarded. Membranerupture pressure is set at 1100 psi and the chamber is evacuated to avacuum of 28 inches of mercury (Hg). The tissue is placed approximately3.5 inches away from the retaining screen and bombarded three times.Following bombardment, the tissue can be divided in half and placed backinto liquid and cultured as described above.

[0101] Five to seven days post bombardment, the liquid media may beexchanged with fresh media, and eleven to twelve days post bombardmentwith fresh media containing 50 mg/mL hygromycin. This selective mediacan be refreshed weekly. Seven to eight weeks post bombardment, green,transformed tissue may be observed growing from untransformed, necroticembryogenic clusters. Isolated green tissue is removed and inoculatedinto individual flasks to generate new, clonally propagated, transformedembryogenic suspension cultures. Each new line may be treated as anindependent transformation event. These suspensions can then besubcultured and maintained as clusters of immature embryos orregenerated into whole plants by maturation and germination ofindividual somatic embryos.

EXAMPLE 6 Expression of Chimeric Genes in Microbial Cells

[0102] The cDNAs encoding the instant polypeptides can be inserted intothe T7 E. coli expression vector pBT430. This vector is a derivative ofpET-3a(Rosenberg et al. (1987) Gene 56:125-135) which employs thebacteriophage T7 RNA polymerase/T7 promoter system. Plasmid pBT430 wasconstructed by first destroying the EcoR I and Hind III sites in pET-3aat their original positions. An oligonucleotide adaptor containing EcoRI and Hind III sites was inserted at the BamH I site of pET-3a. Thiscreated pET-3aM with additional unique cloning sites for insertion ofgenes into the expression vector. Then, the Nde I site at the positionof translation initiation was converted to an Nco I site usingoligonucleotide-directed mutagenesis. The DNA sequence of pET-3aM inthis region, 5′-CATATGG, was converted to 5′-CCCATGG in pBT430.

[0103] Plasmid DNA containing a cDNA may be appropriately digested torelease a nucleic acid fragment encoding the protein. This fragment maythen be purified on a 1% low melting agarose gel. Buffer and agarosecontain 10 μg/mL ethidium bromide for visualization of the DNA fragment.The fragment can then be purified from the agarose gel by digestion withGELase™ (Epicentre Technologies, Madison, Wis.) according to themanufacturer's instructions, ethanol precipitated, dried and resuspendedin 20 μL of water. Appropriate oligonucleotide adapters may be ligatedto the fragment using T4 DNA ligase (New England Biolabs (NEB), Beverly,Mass.). The fragment containing the ligated adapters can be purifiedfrom the excess adapters using low melting agarose as described above.The vector pBT430 is digested, dephosphorylated with alkalinephosphatase (NEB) and deproteinized with phenol/chloroform as describedabove. The prepared vector pBT430 and fragment can then be ligated at16° C. for 15 hours followed by transformation into DH5 electrocompetentcells (GIBCO BRL). Transformants can be selected on agar platescontaining LB media and 100 μg/mL ampicillin. Transformants containingthe gene encoding the instant polypeptides are then screened for thecorrect orientation with respect to the T7 promoter by restrictionenzyme analysis.

[0104] For high level expression, a plasmid clone with the cDNA insertin the correct orientation relative to the T7 promoter can betransformed into E. coli strain BL21 (DE3) (Studier et al. (1986) J.Mol. Biol. 189:113-130). Cultures are grown in LB medium containingampicillin (100 mg/L) at 25° C. At an optical density at 600 nm ofapproximately 1, IPTG (isopropylthio-β-galactoside, the inducer) can beadded to a final concentration of 0.4 mM and incubation can be continuedfor 3 h at 25° C. Cells are then harvested by centrifugation andre-suspended in 50 μL of 50 mM Tris-HCl at pH 8.0 containing 0.1 mM DTTand 0.2 mM phenyl methylsulfonyl fluoride. A small amount of 1 mm glassbeads can be added and the mixture sonicated 3 times for about 5 secondseach time with a microprobe sonicator. The mixture is centrifuged andthe protein concentration of the supernatant determined. One μg ofprotein from the soluble fraction of the culture can be separated bySDS-polyacrylamide gel electrophoresis. Gels can be observed for proteinbands migrating at the expected molecular weight.

1 57 1 859 DNA Zea mays 1 cggagatcca gggctactac gatgacggca ccgccgtggtgtcgttcgac gtgcacaaca 60 tcgacaccac gctgacgaac ttgggcagcg tggtggagtggtggctgggt gagacctatc 120 gcctccacca ccgcggccac atcgccggcc tcgacgtggagtggcgcccc gctcgcgtgc 180 cgggccccgt ccccgtcgcc gtgctgcaga tctgcgtcgaccaccgctgc ctcgtattcc 240 agatcctcca agccgactac atccccgacg ccctgtccaggttcctcgcc gaccgccggt 300 tcaccttcgt gggggtcggg atcagcggcg acgtcgcaaagctgcgggcc gggtacaggc 360 tgggggtggc gagcgccgtg gacctgcgcg ttctcgctgccgacacgctg gaggtgcccg 420 agctgctccg cgcggggctt cagacgctgg tgtgggaggtgatgggcgtg cagatggtga 480 agccgcacca cgtgcgcgtc agcgcctggg acacgcccacgctgtcggaa gaccagctca 540 agtacgcctg cgccgacgct ttcgcctcgt tcgaggtcggccggaggctc tacgaaggcg 600 actactaggg tctagggtta cggtatgctg tctggtttagcaatgccatg cgtgtcaggc 660 cggcgtatca gtattagcag tcgtcgatgg tcttcagtttgcgttgcagt tgtgtcctct 720 aatttctctg cctaataagt tgtactagct agtatcacgcgcgtgatctc ttttgtgtgc 780 gccaccactc gtcatgcata tggcatttcg acttcaaaatttgaatgcta tcttgaagcc 840 caaaaaaaaa aaaaaaaaa 859 2 201 PRT Zea mays 2Glu Ile Gln Gly Tyr Tyr Asp Asp Gly Thr Ala Val Val Ser Phe Asp 1 5 1015 Val His Asn Ile Asp Thr Thr Leu Thr Asn Leu Gly Ser Val Val Glu 20 2530 Trp Trp Leu Gly Glu Thr Tyr Arg Leu His His Arg Gly His Ile Ala 35 4045 Gly Leu Asp Val Glu Trp Arg Pro Ala Arg Val Pro Gly Pro Val Pro 50 5560 Val Ala Val Leu Gln Ile Cys Val Asp His Arg Cys Leu Val Phe Gln 65 7075 80 Ile Leu Gln Ala Asp Tyr Ile Pro Asp Ala Leu Ser Arg Phe Leu Ala 8590 95 Asp Arg Arg Phe Thr Phe Val Gly Val Gly Ile Ser Gly Asp Val Ala100 105 110 Lys Leu Arg Ala Gly Tyr Arg Leu Gly Val Ala Ser Ala Val AspLeu 115 120 125 Arg Val Leu Ala Ala Asp Thr Leu Glu Val Pro Glu Leu LeuArg Ala 130 135 140 Gly Leu Gln Thr Leu Val Trp Glu Val Met Gly Val GlnMet Val Lys 145 150 155 160 Pro His His Val Arg Val Ser Ala Trp Asp ThrPro Thr Leu Ser Glu 165 170 175 Asp Gln Leu Lys Tyr Ala Cys Ala Asp AlaPhe Ala Ser Phe Glu Val 180 185 190 Gly Arg Arg Leu Tyr Glu Gly Asp Tyr195 200 3 968 DNA Zea mays unsure (163) n = A, C, G or T 3 acccacgcgtccgctcttcc ccagttcccc ttgtcgtttg cgcaggggaa aaggagcgcc 60 gcagaccgagggacggcgac gaggtatcca atggccgagc ctgaccctga cgtgatcgaa 120 gtgaccttcggcaacgacgt gattaacacc accgtcacat ccnccggcca ggctgtggag 180 cgctggatcgcggagatcct cgcgttgcac cgccccggca gcaacggcta cagcatcatc 240 gtcgggcttgacgttgagtg gcgcccaagc ttcggcccgc accagaaccc ggtggccaca 300 ctgcagctctgcgtcggaca cagctgcctc atcttccagc tcctctacgc cgactacgtc 360 cccggcgcgctggcggagtt cctcggcgac cgcgggatcc gcttcgtcgg cgtcggcgtg 420 gaggcggacgcggagcggct cagcgacgac cacggtctgg tggtggccaa cgcggaggac 480 ctgcggggccgcgccgcgga gcggatgaac cgcccggacc tccgccaggc ggggctgcgt 540 gcgctcgtgcaagtcgtcat gggcgtcaac ctcgtgaagc cgcagagggt caccatgagc 600 cgctgggacgcgtcctgcct cagctacgag cagatcaagt acgcctgcat cgacgccttc 660 gtctctttcgaggtcgcccg caggctgctt ggcggcgcgt actgatcgac gcggtgaggt 720 gctgtgttgtttaatcctta ctctatcctg tcttagtttg ttgtactttg ccgtggactc 780 ccgttgcttaatccttagcc tatcctatct tagttcgttg cgctttgctt ccagacaatt 840 tgtgctagtgtgctcagact tcagatttgg ttgttgctgc atttcgggct acaaggttgt 900 aggggtttctgtgatcgagg agaattattc agacagtcat gtactgcgtt cctggtttaa 960 aaaaaaaa 9684 234 PRT Zea mays UNSURE (55) Xaa = ANY AMINO ACID 4 Thr His Ala SerAla Leu Pro Gln Phe Pro Leu Ser Phe Ala Gln Gly 1 5 10 15 Lys Arg SerAla Ala Asp Arg Gly Thr Ala Thr Arg Tyr Pro Met Ala 20 25 30 Glu Pro AspPro Asp Val Ile Glu Val Thr Phe Gly Asn Asp Val Ile 35 40 45 Asn Thr ThrVal Thr Ser Xaa Gly Gln Ala Val Glu Arg Trp Ile Ala 50 55 60 Glu Ile LeuAla Leu His Arg Pro Gly Ser Asn Gly Tyr Ser Ile Ile 65 70 75 80 Val GlyLeu Asp Val Glu Trp Arg Pro Ser Phe Gly Pro His Gln Asn 85 90 95 Pro ValAla Thr Leu Gln Leu Cys Val Gly His Ser Cys Leu Ile Phe 100 105 110 GlnLeu Leu Tyr Ala Asp Tyr Val Pro Gly Ala Leu Ala Glu Phe Leu 115 120 125Gly Asp Arg Gly Ile Arg Phe Val Gly Val Gly Val Glu Ala Asp Ala 130 135140 Glu Arg Leu Ser Asp Asp His Gly Leu Val Val Ala Asn Ala Glu Asp 145150 155 160 Leu Arg Gly Arg Ala Ala Glu Arg Met Asn Arg Pro Asp Leu ArgGln 165 170 175 Ala Gly Leu Arg Ala Leu Val Gln Val Val Met Gly Val AsnLeu Val 180 185 190 Lys Pro Gln Arg Val Thr Met Ser Arg Trp Asp Ala SerCys Leu Ser 195 200 205 Tyr Glu Gln Ile Lys Tyr Ala Cys Ile Asp Ala PheVal Ser Phe Glu 210 215 220 Val Ala Arg Arg Leu Leu Gly Gly Ala Tyr 225230 5 2277 DNA Zea mays unsure (1186) n = A, C, G or T 5 gcgaccggcgcctagcgttc tgtggccgcc gcccttctgc cgtccgaccg gaccaggtcg 60 ccgacgtcgcccctatcccg cacgcgtcat cgcagcgcct ccgagtctcc gccaagatct 120 gcctcggccacggcgcggcg ccgctggagc tgcagccgtc cagccgactg ccttccgccg 180 accaccgaccaccgaccact ggccgagccg ccgcagctag ctgcgctttg aagcatgggt 240 cgttttgaacaagttactcc agaaggccct gaaactaatc agcatgatga gcagcgctcc 300 atacgtttacatgcattttc tgatctatca cacgtccctg cagccacttt tatttatctc 360 ttaaaggactgctatggata tggtacgaac aaagcaacct caaagtttaa gatcctcatg 420 cagctggtaaaagtggcatt gcataatggt ccacagccag gcccgttcac atatgttgtc 480 cagtgtatgtatattgtacc tctgctgggg aaaacttatt ctgaagggtt cagccatatg 540 ttgacatcctctttaaagca cttgaaatct gtggaatcag cacagaaaga tttcttggag 600 gcaaagcaccttgccgcaca acttgttctt gatatccttg attctattgt accccatgag 660 aaccgcatattggtcaagct tcttgagaca tttgaaattg agttgagaga catggcccgt 720 gctttgtatgattcagagtt ggacgatggt gatctaatga aagcacgtga acatctcaga 780 cagcaagttaagcgctgtat ggaatcagaa tccaatgcac ttgctgtgac tctaataaca 840 catttttccatccaatgctg tgatgagtcc ttcatcataa aattgattaa aaacaatcaa 900 ttagagattgcagagcagtg tgctattttc atgggcaagg aaatgatatc gctagtcgtt 960 caaaagtatctagatatgaa aatgctgaag agtgcaaaca aattggtcaa agaacatgaa 1020 ctcacagaagagtttccaga tgttagctat ttgtataaag agagttcagt aaagaagttg 1080 gctgagaaaggatgctggga tattgcagaa actagggcca agaaggacac aaaactcctg 1140 gaatacctggtatatttagc aatggaagct ggttatatgg agaagnttga cgagctttgc 1200 aagcgatactctattgaagg ttatgttgat tctttggttc cggagaaggt tttctgtgta 1260 tctgactacttagatctgaa gaaattggat gtggaagaaa ttgtctgggt tgatgagatc 1320 aatgggctgcttaatgcaac aagtgatatt gaagcttgta aaattattgg catggattgt 1380 gagtggagacctaatttcga gaaaaatact aaatctagta aggtctcaat catacaaatt 1440 gcatcagataagatagcctt catttttgat ctgattaaac tgtatgaaga tgacccaaaa 1500 gcattggacagttgcttgag gcgtgttatg tgttcatcta agatactaaa gctgggctat 1560 gacattcagtgtgatcttca tcaactaacg cgatcatacg gagaattgga atgttttcag 1620 tcctatgaaatggtacttga tatgcagaag cttttcaaag gcgttactgg tggcctctct 1680 ggattgtcaaaggaaatatt aggagctggt ttgaacaaga cccggcgaaa tagcaactgg 1740 gagcaacggccactaaccta aaatcagaaa gagtacgctg ctcttgatgc cgtggtcctt 1800 gtgcatatattccatgaaca catgcggagg caagcacagt tcggtgtctc tgagggaagc 1860 agagtcgagtggaggtctca cgttgtttcc cgagtgagtt gcacgcgtac gcccttgcgt 1920 ttctagtggtggggttggac agcttggagt gaaattactt ctagcatctg catgtgccgt 1980 cttctaacatctatgatgac agttctctgg catggcatca taggctcaca gttcatcccc 2040 agaagttagtgctgcgactt cgttgctctt gccttgtgta tatacagtat attgttacct 2100 ccaccggagtttttcctttc ctgctgtatc gcattcagtg ctgagtgcat tattagattt 2160 ggaacccgttttctgttatg aattagtgat aatggaagtc aggaagtgtc gcgttaaaaa 2220 aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaag 2277 6 563 PRT Zeamays UNSURE (318) Xaa = ANY AMINO ACID 6 Met Gly Arg Phe Glu Gln Val ThrPro Glu Gly Pro Glu Thr Asn Gln 1 5 10 15 His Asp Glu Gln Arg Ser IleArg Leu His Ala Phe Ser Asp Leu Ser 20 25 30 His Val Pro Ala Ala Thr PheIle Tyr Leu Leu Lys Asp Cys Tyr Gly 35 40 45 Tyr Gly Thr Asn Lys Ala ThrSer Lys Phe Lys Ile Leu Met Gln Leu 50 55 60 Val Lys Val Ala Leu His AsnGly Pro Gln Pro Gly Pro Phe Thr Tyr 65 70 75 80 Val Val Gln Cys Met TyrIle Val Pro Leu Leu Gly Lys Thr Tyr Ser 85 90 95 Glu Gly Phe Ser His MetLeu Thr Ser Ser Leu Lys His Leu Lys Ser 100 105 110 Val Glu Ser Ala GlnLys Asp Phe Leu Glu Ala Lys His Leu Ala Ala 115 120 125 Gln Leu Val LeuAsp Ile Leu Asp Ser Ile Val Pro His Glu Asn Arg 130 135 140 Ile Leu ValLys Leu Leu Glu Thr Phe Glu Ile Glu Leu Arg Asp Met 145 150 155 160 AlaArg Ala Leu Tyr Asp Ser Glu Leu Asp Asp Gly Asp Leu Met Lys 165 170 175Ala Arg Glu His Leu Arg Gln Gln Val Lys Arg Cys Met Glu Ser Glu 180 185190 Ser Asn Ala Leu Ala Val Thr Leu Ile Thr His Phe Ser Ile Gln Cys 195200 205 Cys Asp Glu Ser Phe Ile Ile Lys Leu Ile Lys Asn Asn Gln Leu Glu210 215 220 Ile Ala Glu Gln Cys Ala Ile Phe Met Gly Lys Glu Met Ile SerLeu 225 230 235 240 Val Val Gln Lys Tyr Leu Asp Met Lys Met Leu Lys SerAla Asn Lys 245 250 255 Leu Val Lys Glu His Glu Leu Thr Glu Glu Phe ProAsp Val Ser Tyr 260 265 270 Leu Tyr Lys Glu Ser Ser Val Lys Lys Leu AlaGlu Lys Gly Cys Trp 275 280 285 Asp Ile Ala Glu Thr Arg Ala Lys Lys AspThr Lys Leu Leu Glu Tyr 290 295 300 Leu Val Tyr Leu Ala Met Glu Ala GlyTyr Met Glu Lys Xaa Asp Glu 305 310 315 320 Leu Cys Lys Arg Tyr Ser IleGlu Gly Tyr Val Asp Ser Leu Val Pro 325 330 335 Glu Lys Val Phe Cys ValSer Asp Tyr Leu Asp Leu Lys Lys Leu Asp 340 345 350 Val Glu Glu Ile ValTrp Val Asp Glu Ile Asn Gly Leu Leu Asn Ala 355 360 365 Thr Ser Asp IleGlu Ala Cys Lys Ile Ile Gly Met Asp Cys Glu Trp 370 375 380 Arg Pro AsnPhe Glu Lys Asn Thr Lys Ser Ser Lys Val Ser Ile Ile 385 390 395 400 GlnIle Ala Ser Asp Lys Ile Ala Phe Ile Phe Asp Leu Ile Lys Leu 405 410 415Tyr Glu Asp Asp Pro Lys Ala Leu Asp Ser Cys Leu Arg Arg Val Met 420 425430 Cys Ser Ser Lys Ile Leu Lys Leu Gly Tyr Asp Ile Gln Cys Asp Leu 435440 445 His Gln Leu Thr Arg Ser Tyr Gly Glu Leu Glu Cys Phe Gln Ser Tyr450 455 460 Glu Met Val Leu Asp Met Gln Lys Leu Phe Lys Gly Val Thr GlyGly 465 470 475 480 Leu Ser Gly Leu Ser Lys Glu Ile Leu Gly Ala Gly LeuAsn Lys Thr 485 490 495 Arg Arg Asn Ser Asn Trp Glu Gln Arg Pro Leu ThrXaa Asn Gln Lys 500 505 510 Glu Tyr Ala Ala Leu Asp Ala Val Val Leu ValHis Ile Phe His Glu 515 520 525 His Met Arg Arg Gln Ala Gln Phe Gly ValSer Glu Gly Ser Arg Val 530 535 540 Glu Trp Arg Ser His Val Val Ser ArgVal Ser Cys Thr Arg Thr Pro 545 550 555 560 Leu Arg Phe 7 1275 DNA Zeamays 7 ccacgcgtcc gaggtggagc ctttcttgga tgtcaccaac atttattact atctcaaggg60 ccatgatagg cagaagaagc ttccaaagga gaccaagagt ttggcaacta tttgtgagga 120gctgcttggt atccttttgt ccaaggaact ccagtgtagt gattggtcat gccgcccctt 180aagtgaaggg caaatacaat atgctgcgtc ggatgcctac tacttgctag acatatttga 240tttgttccaa aaaaggatca caatggaagg aaaatgttca tctacaacag aacttacttc 300agacaggcat tgctcatcag tggtgataga atgctcttct tctggatatg gcatttgctc 360gggtagttgt ttgatgtcca tagtaaccaa gtacagtgag aagataatat tgacagaatc 420tgatgcaaaa ccgcgtacct ccagacgaaa agaaaaactg aagattcctg ccaatgccaa 480acgcaaagat aatgtggatt gcagtagtga atggcagggt ccccctccat gggatccttc 540cattggtggg gatgggtacc caaagttctt gtgtgatgtg atgattgagg gcctagctaa 600gcacttgcga tgtgttggaa tagatgctgc aattccatct tcaaagaaac ctgaaccaag 660ggatctatta aatcaaacat acaaggaagg aagaatatta ttaacacggg atgccaagct 720cttgaaatat caatatttag ctggtaacca ggtcattaat atcttccaat taaagatttc 780tgaggaccaa cttatgtcga ggtgcacaaa atgcaatggt agctttattc agaaaccact 840taccctagag gaagctgttg aagcctcaaa aggtttccag attattccca cgtgcttgtt 900caaccgaaat ctggagttct ggaagtgcac cgattgcaac caactctact gggaggggac 960tcagtaccac aatgcagtcc aaaggttctt gtcagtgtgc aacattagtg actaagcagc 1020acgtacggcc ttggtactca tttgtaaaat tgtaaggtac tgagatacca tcacctccga 1080aagatcgaaa ataatctgat ctttggcatc cactcgtgca cgatgaggca tgccatccat 1140ttttagtggt ttgctgttcg tcactgagat gctgttgtaa ggaaaataga ccttgactta 1200tttgattaat ggactttgct ccatgcacat ccaagaggaa aggttctaga tacgtaaaaa 1260aaaaaaaaaa aaaag 1275 8 336 PRT Zea mays 8 Ala Ser Glu Val Glu Pro PheLeu Asp Val Thr Asn Ile Tyr Tyr Tyr 1 5 10 15 Leu Lys Gly His Asp ArgGln Lys Lys Leu Pro Lys Glu Thr Lys Ser 20 25 30 Leu Ala Thr Ile Cys GluGlu Leu Leu Gly Ile Leu Leu Ser Lys Glu 35 40 45 Leu Gln Cys Ser Asp TrpSer Cys Arg Pro Leu Ser Glu Gly Gln Ile 50 55 60 Gln Tyr Ala Ala Ser AspAla Tyr Tyr Leu Leu Asp Ile Phe Asp Leu 65 70 75 80 Phe Gln Lys Arg IleThr Met Glu Gly Lys Cys Ser Ser Thr Thr Glu 85 90 95 Leu Thr Ser Asp ArgHis Cys Ser Ser Val Val Ile Glu Cys Ser Ser 100 105 110 Ser Gly Tyr GlyIle Cys Ser Gly Ser Cys Leu Met Ser Ile Val Thr 115 120 125 Lys Tyr SerGlu Lys Ile Ile Leu Thr Glu Ser Asp Ala Lys Pro Arg 130 135 140 Thr SerArg Arg Lys Glu Lys Leu Lys Ile Pro Ala Asn Ala Lys Arg 145 150 155 160Lys Asp Asn Val Asp Cys Ser Ser Glu Trp Gln Gly Pro Pro Pro Trp 165 170175 Asp Pro Ser Ile Gly Gly Asp Gly Tyr Pro Lys Phe Leu Cys Asp Val 180185 190 Met Ile Glu Gly Leu Ala Lys His Leu Arg Cys Val Gly Ile Asp Ala195 200 205 Ala Ile Pro Ser Ser Lys Lys Pro Glu Pro Arg Asp Leu Leu AsnGln 210 215 220 Thr Tyr Lys Glu Gly Arg Ile Leu Leu Thr Arg Asp Ala LysLeu Leu 225 230 235 240 Lys Tyr Gln Tyr Leu Ala Gly Asn Gln Val Ile AsnIle Phe Gln Leu 245 250 255 Lys Ile Ser Glu Asp Gln Leu Met Ser Arg CysThr Lys Cys Asn Gly 260 265 270 Ser Phe Ile Gln Lys Pro Leu Thr Leu GluGlu Ala Val Glu Ala Ser 275 280 285 Lys Gly Phe Gln Ile Ile Pro Thr CysLeu Phe Asn Arg Asn Leu Glu 290 295 300 Phe Trp Lys Cys Thr Asp Cys AsnGln Leu Tyr Trp Glu Gly Thr Gln 305 310 315 320 Tyr His Asn Ala Val GlnArg Phe Leu Ser Val Cys Asn Ile Ser Asp 325 330 335 9 669 DNA Zea mays 9ccacgcgtcc gcggacgcgt gggtcggggt taggagatct gacccaatgg ccgcgaccaa 60ggtgtgcaac gttaggttcg agggcaacgt gatcaccacc accgtgacgg cctccggcgc 120ggccgtggag agctggctcg acgagatcct ctccgtccac cgccgccgcc tgcacaagct 180cgtcgtcggg ctggacgtcg agtggcgccc cagcttcagc cgcgcctaca gcaaaacagc 240catcgtccag ctctgcgtcg ggcgccgctg cctcatcttc cagcttctcc acgccgacta 300cgtccccaac acgctggatg agttcctcag cgaccccgac tacacattcg tcggcgtggg 360cgtggctgcg gacgtcgagc ggctcgagaa cgactacgac ctggaggtgg cgaacgcgga 420ggacctggcc gaactcgcgg ccaaggagat ggggcgcccg gacctccgca acgcgggcct 480gcagggcatc gcgagagccg tcatggacgc ccacgtcgag aagccgcagt gggtgaggac 540gggcccctgg gacgcgtcat ccctctccga cgagcagatc gagtacgcca ccatcgacgc 600ttttgtctct ttcgaggttg gccggatgct gctcagcggc tactattgat cgatcgagcc 660gttaatgca 669 10 213 PRT Zea mays 10 Ser Ala Asp Ala Trp Val Gly Val ArgArg Ser Asp Pro Met Ala Ala 1 5 10 15 Thr Lys Val Cys Asn Val Arg PheGlu Gly Asn Val Ile Thr Thr Thr 20 25 30 Val Thr Ala Ser Gly Ala Ala ValGlu Ser Trp Leu Asp Glu Ile Leu 35 40 45 Ser Val His Arg Arg Arg Leu HisLys Leu Val Val Gly Leu Asp Val 50 55 60 Glu Trp Arg Pro Ser Phe Ser ArgAla Tyr Ser Lys Thr Ala Ile Val 65 70 75 80 Gln Leu Cys Val Gly Arg ArgCys Leu Ile Phe Gln Leu Leu His Ala 85 90 95 Asp Tyr Val Pro Asn Thr LeuAsp Glu Phe Leu Ser Asp Pro Asp Tyr 100 105 110 Thr Phe Val Gly Val GlyVal Ala Ala Asp Val Glu Arg Leu Glu Asn 115 120 125 Asp Tyr Asp Leu GluVal Ala Asn Ala Glu Asp Leu Ala Glu Leu Ala 130 135 140 Ala Lys Glu MetGly Arg Pro Asp Leu Arg Asn Ala Gly Leu Gln Gly 145 150 155 160 Ile AlaArg Ala Val Met Asp Ala His Val Glu Lys Pro Gln Trp Val 165 170 175 ArgThr Gly Pro Trp Asp Ala Ser Ser Leu Ser Asp Glu Gln Ile Glu 180 185 190Tyr Ala Thr Ile Asp Ala Phe Val Ser Phe Glu Val Gly Arg Met Leu 195 200205 Leu Ser Gly Tyr Tyr 210 11 776 DNA Oryza sativa 11 cgacgacgcgccacacggtc cgcttcggct ccgccacgat cgacacgacg gtcaccagcg 60 acgtcgcggccgccgacgag tgggcgcgcg gcgtccgcgc cgcggcgagg ggcggccgcg 120 gcctgatcgtcggcctcgac tgcgagtgga agcccaacca cgtctcctgg aagacctcca 180 aggtggccgtcctccagctc tgcgccggcg agcgcttctg cctcgtcctg cagctgttct 240 acgccaaccgcgtcccgccc gccgtcgcgg acctcctcgg cgacccgtcc gtgcggctcg 300 tcggcatcggcgtcggcgag gacgcggcga agctggaggc cgactacggc gtctggtgcg 360 ccgcgccggtggacctggag gacgcctgca accgccggct cggcctcgtc gggaccggga 420 ggaggctggggctgaagggc tacgcgaggg aggtgctcgg gatggccatg gagaagccga 480 ggcgcgtaaccatgagcaac tgggagaagc gggagctgga cccggcgcag gtcgagtacg 540 cctgcatcgacgcctacgtt tcctacaagc tgggcgagag ggtccttgcc aactgatcat 600 gatgcagtgcaactatggaa ctatccatgc acaacagcac aagtggagta gtagttttct 660 ttgttgctttggtcacccct gtattcagtg gtgctatata tgttagccaa tcgttctaac 720 ctggaatgctattctagttt gttgtttcat caaaaaaaaa aaaaaaaaac tcgtgc 776 12 197 PRT Oryzasativa 12 Thr Thr Arg His Thr Val Arg Phe Gly Ser Ala Thr Ile Asp ThrThr 1 5 10 15 Val Thr Ser Asp Val Ala Ala Ala Asp Glu Trp Ala Arg GlyVal Arg 20 25 30 Ala Ala Ala Arg Gly Gly Arg Gly Leu Ile Val Gly Leu AspCys Glu 35 40 45 Trp Lys Pro Asn His Val Ser Trp Lys Thr Ser Lys Val AlaVal Leu 50 55 60 Gln Leu Cys Ala Gly Glu Arg Phe Cys Leu Val Leu Gln LeuPhe Tyr 65 70 75 80 Ala Asn Arg Val Pro Pro Ala Val Ala Asp Leu Leu GlyAsp Pro Ser 85 90 95 Val Arg Leu Val Gly Ile Gly Val Gly Glu Asp Ala AlaLys Leu Glu 100 105 110 Ala Asp Tyr Gly Val Trp Cys Ala Ala Pro Val AspLeu Glu Asp Ala 115 120 125 Cys Asn Arg Arg Leu Gly Leu Val Gly Thr GlyArg Arg Leu Gly Leu 130 135 140 Lys Gly Tyr Ala Arg Glu Val Leu Gly MetAla Met Glu Lys Pro Arg 145 150 155 160 Arg Val Thr Met Ser Asn Trp GluLys Arg Glu Leu Asp Pro Ala Gln 165 170 175 Val Glu Tyr Ala Cys Ile AspAla Tyr Val Ser Tyr Lys Leu Gly Glu 180 185 190 Arg Val Leu Ala Asn 19513 1006 DNA Glycine max 13 gcaccagttt aggttcaaat ccgcctccac actcaatcgaaatccgaaaa ttccattgtt 60 tccattgaaa cacggtttct ccaaaatcaa aaccccctttcttctctcac cttctccaat 120 tccccgtcat gatatcatcg cggcaagacc aaaccggcgcgccgctcagc tcctccacca 180 ccaccaccac ccccgtggtg gcgccgatca gcgtggtggaccacggcctc ccctacgaca 240 cccacaacct ctacgacgtc tccttcaaca acacccacaccatctacacc ctcctcacct 300 ccgacccctc cctcgtcgac tcctggatct ccaccgtcctccgcgaccac cagcagcgcg 360 tcctcaccgt gggcctcgac atcgagtggc gccccaacacccagcgcaac atgcagaacc 420 ccgtggccac actccagctc tgcgtcgccg aacgctgcctcgtcttccag attctccact 480 ccccttcaat ccctccctct cttgtttcct tcctcgctgaccctaacatc actttcgttg 540 gtgttgggat ccaagaagac gtggagaagc ttctagaagattataatctt aacgtggcga 600 atgttcgtga ccttcgctcc ttcgctgcgg agaggcttggcgaccttgag ctgaaacggg 660 ccgggctcaa gtctttgggc ctccgcgtgc tgggcctggaagttgccaag cccaagcggg 720 tcaccaggag taggtgggac aatccctggc tcactgcccagcaggttcag tatgcagccg 780 ttgatgcctt tctctcttac gagattgatc gccgtttgagttcttataat taattcatgc 840 cttttgtttt tttttccttt tctcacttgt cttgtgtaggagtatttggt aattttgtga 900 attgtaacac ttggtgttgc ttgctatgtt accattcgaatgcttaattt gatttttctg 960 aaatttggac cttggttctg atacaaaaaa aaaaaaaaaaaaaaaa 1006 14 234 PRT Glycine max 14 Met Ile Ser Ser Arg Gln Asp GlnThr Gly Ala Pro Leu Ser Ser Ser 1 5 10 15 Thr Thr Thr Thr Thr Pro ValVal Ala Pro Ile Ser Val Val Asp His 20 25 30 Gly Leu Pro Tyr Asp Thr HisAsn Leu Tyr Asp Val Ser Phe Asn Asn 35 40 45 Thr His Thr Ile Tyr Thr LeuLeu Thr Ser Asp Pro Ser Leu Val Asp 50 55 60 Ser Trp Ile Ser Thr Val LeuArg Asp His Gln Gln Arg Val Leu Thr 65 70 75 80 Val Gly Leu Asp Ile GluTrp Arg Pro Asn Thr Gln Arg Asn Met Gln 85 90 95 Asn Pro Val Ala Thr LeuGln Leu Cys Val Ala Glu Arg Cys Leu Val 100 105 110 Phe Gln Ile Leu HisSer Pro Ser Ile Pro Pro Ser Leu Val Ser Phe 115 120 125 Leu Ala Asp ProAsn Ile Thr Phe Val Gly Val Gly Ile Gln Glu Asp 130 135 140 Val Glu LysLeu Leu Glu Asp Tyr Asn Leu Asn Val Ala Asn Val Arg 145 150 155 160 AspLeu Arg Ser Phe Ala Ala Glu Arg Leu Gly Asp Leu Glu Leu Lys 165 170 175Arg Ala Gly Leu Lys Ser Leu Gly Leu Arg Val Leu Gly Leu Glu Val 180 185190 Ala Lys Pro Lys Arg Val Thr Arg Ser Arg Trp Asp Asn Pro Trp Leu 195200 205 Thr Ala Gln Gln Val Gln Tyr Ala Ala Val Asp Ala Phe Leu Ser Tyr210 215 220 Glu Ile Asp Arg Arg Leu Ser Ser Tyr Asn 225 230 15 1170 DNAGlycine max 15 gcacgagagg agggtggttt tacgcgactt gcctcattcg tctgcgattgcgagcctttg 60 acggaggatg atctggaagc catcgaagcc tctctttcca ataacaaaaaacgtccattc 120 aacgatcaca ctcacactcc tcgtcgtcgc ttgcccaaat cgcttatcgctcttcaacac 180 ccaaacgctt cttctttctc gccccatccc cgtccatgcg attcaagaatgacattgcct 240 gtaatgaagt ttagtggtca aatttcttat agcaggactt ttgatgctgtagagaaagct 300 gcaacaaagc tcttacaaat tctccaagaa aaaacgaccg acatgatgcaaactgcaatt 360 ggatttgaca ttgagtggaa acccaccttc agaaaaggtg ttcctcccggaaaggtagca 420 gtgatgcaga tatgtggtga cactagacat tgtcatgttc tacatctaattcattctgga 480 atccctcaaa atttacagct tttgcttgaa gatcccacag tcttgaaggttggagctggg 540 attgatggtg atgctgtgaa ggtttttaga gattataaca tatctgttaaaggtgtgacg 600 gatctttctt ttcatgctaa tcaaaagctt ggtggagatc ataagtggggtcttgcatct 660 ttgactgaaa aacttctatc aaaacagctt aaaaagccca acaaaataagactgggaaat 720 tgggaggctc ctgttttgtc aaaggagcaa ctagagtatg ctgcaacagatgcttttgct 780 tcttggtgtc tttatcaggc gattaaagat ctcccggacg cccagaaagtcactgacaga 840 agtggccaag ttgatgctgt accgcaagaa tgactacatc tttagtgtgtattgaatttc 900 atttctatta aatattaatc atttgtcatg tactaattat ctgttgtaatctgtaaaaat 960 aaaaaatctg ttgtaacatt ttatttttgc tgcttggaca cccataggagagtgaaaatc 1020 cccttttggg agtatgataa gaaaatagaa aggaatagat aaaatggctgatgtgattaa 1080 aaaacgatgg acatgatgtc tattgggcgt agaatttgag gtgtataaaataaatttttt 1140 ggaataaaaa aaaaaaaaaa aaaaaaaaaa 1170 16 290 PRT Glycinemax 16 Ala Arg Glu Glu Gly Gly Phe Thr Arg Leu Ala Ser Phe Val Cys Asp 15 10 15 Cys Glu Pro Leu Thr Glu Asp Asp Leu Glu Ala Ile Glu Ala Ser Leu20 25 30 Ser Asn Asn Lys Lys Arg Pro Phe Asn Asp His Thr His Thr Pro Arg35 40 45 Arg Arg Leu Pro Lys Ser Leu Ile Ala Leu Gln His Pro Asn Ala Ser50 55 60 Ser Phe Ser Pro His Pro Arg Pro Cys Asp Ser Arg Met Thr Leu Pro65 70 75 80 Val Met Lys Phe Ser Gly Gln Ile Ser Tyr Ser Arg Thr Phe AspAla 85 90 95 Val Glu Lys Ala Ala Thr Lys Leu Leu Gln Ile Leu Gln Glu LysThr 100 105 110 Thr Asp Met Met Gln Thr Ala Ile Gly Phe Asp Ile Glu TrpLys Pro 115 120 125 Thr Phe Arg Lys Gly Val Pro Pro Gly Lys Val Ala ValMet Gln Ile 130 135 140 Cys Gly Asp Thr Arg His Cys His Val Leu His LeuIle His Ser Gly 145 150 155 160 Ile Pro Gln Asn Leu Gln Leu Leu Leu GluAsp Pro Thr Val Leu Lys 165 170 175 Val Gly Ala Gly Ile Asp Gly Asp AlaVal Lys Val Phe Arg Asp Tyr 180 185 190 Asn Ile Ser Val Lys Gly Val ThrAsp Leu Ser Phe His Ala Asn Gln 195 200 205 Lys Leu Gly Gly Asp His LysTrp Gly Leu Ala Ser Leu Thr Glu Lys 210 215 220 Leu Leu Ser Lys Gln LeuLys Lys Pro Asn Lys Ile Arg Leu Gly Asn 225 230 235 240 Trp Glu Ala ProVal Leu Ser Lys Glu Gln Leu Glu Tyr Ala Ala Thr 245 250 255 Asp Ala PheAla Ser Trp Cys Leu Tyr Gln Ala Ile Lys Asp Leu Pro 260 265 270 Asp AlaGln Lys Val Thr Asp Arg Ser Gly Gln Val Asp Ala Val Pro 275 280 285 GlnGlu 290 17 1081 DNA Vernonia mespilifolia 17 gcacgagtaa taattctactagaatattaa gaaccacgtt ttgtggagca gtagagatgg 60 tcagagcaac tacagttgaataccgtcaaa gactggatgc tgaacaggga gctagttcat 120 ggaaatcaag tggggatgctataccgatgg attacgcgct gttgcagatt atcagagaat 180 atggtgataa aatagtattgacggagactg atggaaagcc aagatcatca aaaaaaaaag 240 gaaaaatgaa gtcctccaatgggtttgcat gcaaaggaaa acaagtagat gatatagacg 300 aatggcaagg cccagcaccatgggaaaatt tgttgggtgg tgatggattg ccaaagtttc 360 tgtgtgatgt gatggtggagggcctagcaa aacatttaag gtgtgttgga attgatgctg 420 ctgttcccta ttccaaaaagcctgaagcca gggacttgat tgatcaagct attaaagaga 480 agagagttat cctgacaagagatgccaagc tcttaagaca tgaatatctg ttaaaaaatc 540 agatatataa agtgaagagtcttctgaaaa atgaccagct aaatgaggtg atagagactt 600 ttgagttgaa gatttgcgaggatcaactga tgtcaaggtg cactaaatgc aatgggaggt 660 tcatccagaa accactgtcaattgaagagg ccattgaagc agcaaaagga tttcaagtga 720 tcccaaactg tttgtttgacaggaacatcg agttttggca gtgcacagat tgcaatcaac 780 tctactggga gggaacgcagtaccacaatg cggtacagaa attcattgac gtttgtaaga 840 taaacccaac tgtgtgaaggtgctgacggc cctctgccct ttctcctatt ttatttttga 900 ccatcaaagc tttacttcaaaaaaaaaaaa tttttttttt taccccaatc atagtaccat 960 tacctctcat aaataatgaacacataatat ggtcaatcga caattatgat tcaatgtctt 1020 tcgtacccac tcatctcaataaaaatattg ttttcaaaaa aaaaaaaaaa aaaaaaaaaa 1080 a 1081 18 284 PRTVernonia mespilifolia 18 Thr Ser Asn Asn Ser Thr Arg Ile Leu Arg Thr ThrPhe Cys Gly Ala 1 5 10 15 Val Glu Met Val Arg Ala Thr Thr Val Glu TyrArg Gln Arg Leu Asp 20 25 30 Ala Glu Gln Gly Ala Ser Ser Trp Lys Ser SerGly Asp Ala Ile Pro 35 40 45 Met Asp Tyr Ala Leu Leu Gln Ile Ile Arg GluTyr Gly Asp Lys Ile 50 55 60 Val Leu Thr Glu Thr Asp Gly Lys Pro Arg SerSer Lys Lys Lys Gly 65 70 75 80 Lys Met Lys Ser Ser Asn Gly Phe Ala CysLys Gly Lys Gln Val Asp 85 90 95 Asp Ile Asp Glu Trp Gln Gly Pro Ala ProTrp Glu Asn Leu Leu Gly 100 105 110 Gly Asp Gly Leu Pro Lys Phe Leu CysAsp Val Met Val Glu Gly Leu 115 120 125 Ala Lys His Leu Arg Cys Val GlyIle Asp Ala Ala Val Pro Tyr Ser 130 135 140 Lys Lys Pro Glu Ala Arg AspLeu Ile Asp Gln Ala Ile Lys Glu Lys 145 150 155 160 Arg Val Ile Leu ThrArg Asp Ala Lys Leu Leu Arg His Glu Tyr Leu 165 170 175 Leu Lys Asn GlnIle Tyr Lys Val Lys Ser Leu Leu Lys Asn Asp Gln 180 185 190 Leu Asn GluVal Ile Glu Thr Phe Glu Leu Lys Ile Cys Glu Asp Gln 195 200 205 Leu MetSer Arg Cys Thr Lys Cys Asn Gly Arg Phe Ile Gln Lys Pro 210 215 220 LeuSer Ile Glu Glu Ala Ile Glu Ala Ala Lys Gly Phe Gln Val Ile 225 230 235240 Pro Asn Cys Leu Phe Asp Arg Asn Ile Glu Phe Trp Gln Cys Thr Asp 245250 255 Cys Asn Gln Leu Tyr Trp Glu Gly Thr Gln Tyr His Asn Ala Val Gln260 265 270 Lys Phe Ile Asp Val Cys Lys Ile Asn Pro Thr Val 275 280 19616 DNA Triticum aestivum 19 ggaattatta gatcaaatct acagggaagg aagaatattactaacacgag atgccaaact 60 cataaagtat gagtatttgg cgactaatca agtatacagagtgaaaagcc tgctcaaaca 120 tgatcaactg gctgaggtat gtgcttattt tgatattttccatcttcagg accgattaat 180 gtcaagatgt acaaagtgca acggaagttt cattcagaaaccattaacgc tcgaggaagc 240 catagaagcc tccaaaggtt ttcaggtcat cccctcatgcctcttcaacc gaaatatgga 300 gttctggaag tgcactgact gcaaccaact ctactgggagggaactcagt accacaacgc 360 agttcaaaaa ttcatgtcag tctgcaacat tagtgagtgagcattgtaac atttcattct 420 caagacgctc atgttgtttt gtatgtgaaa ttcaacatgtagtaggaaac tgctctttca 480 ataatgtcac catctatgcc ttcggacaaa tcccagttgcaattttactt tatgatgtac 540 agtctacagt atttcatatg gtagtaacac atatgcaaatcatatggtag taacatatat 600 acattgatta ccgaca 616 20 133 PRT Triticumaestivum 20 Arg Glu Leu Leu Asp Gln Ile Tyr Arg Glu Gly Arg Ile Leu LeuThr 1 5 10 15 Arg Asp Ala Lys Leu Ile Lys Tyr Glu Tyr Leu Ala Thr AsnGln Val 20 25 30 Tyr Arg Val Lys Ser Leu Leu Lys His Asp Gln Leu Ala GluVal Cys 35 40 45 Ala Tyr Phe Asp Ile Phe His Leu Gln Asp Arg Leu Met SerArg Cys 50 55 60 Thr Lys Cys Asn Gly Ser Phe Ile Gln Lys Pro Leu Thr LeuGlu Glu 65 70 75 80 Ala Ile Glu Ala Ser Lys Gly Phe Gln Val Ile Pro SerCys Leu Phe 85 90 95 Asn Arg Asn Met Glu Phe Trp Lys Cys Thr Asp Cys AsnGln Leu Tyr 100 105 110 Trp Glu Gly Thr Gln Tyr His Asn Ala Val Gln LysPhe Met Ser Val 115 120 125 Cys Asn Ile Ser Glu 130 21 851 DNA Triticumaestivum unsure (376)..(377)..(378) n = A, C, G or T 21 tctacctagccgtctaatca acttcctgca cgtcacttct cccatttact tctccatcgg 60 gaaccgattctcctcggctt cttcacctcc ccttcgtctt catttgcatc caaaccccac 120 tgcgttcacattgttttaac ctctttaaat aatcatctac tacttcagtt agactagcca 180 tctaatcctaattctccaaa ccatggccct ccattccagc ggttccaaat ggggaaagag 240 atcgagatgcaggtttttag cgtccaacat gtcctcgtcg aaggctcgat gcgcataatt 300 gccaccgtcaccgaccaccc aagggttgtt cggcagtggt ttaacaatgg ttccaactcc 360 ctccaaaaggtagagnnnag ggtcaccggt ctcgacgcag agtacacgga gcgtgcccct 420 gggaagatccagcgcaccgc cgtccttcag ctgtgcctca aagatgatgt tttggtgtac 480 cacatcatacactcgccctc cataccaggt gagcttcacg atttcttgtc tcgggaggac 540 gtatacttttatggggcagc catcaaaggg gacaaacaga agctggagcc gtacaacctt 600 gatttgaaaagcatcgctga cctacaaact agaatcaaaa ttcctgtaga agattgtgat 660 aaacagacaccatccctatt cgacgtagca aattttgtgc tcggtacaaa tcttcagaag 720 ggtgacgagccggtggcatt aaggtcgtct ggatgggaga attatccttt gacgtacgag 780 cggataaaataagctgccct cgatgcctgt gtgagtttcg agatagctgc aaggtccaaa 840 gagcttgttg c851 22 192 PRT Triticum aestivum UNSURE (50) Xaa = ANY AMINO ACID 22 MetGly Lys Glu Ile Glu Met Gln Val Phe Ser Val Gln His Val Leu 1 5 10 15Val Glu Gly Ser Met Arg Ile Ile Ala Thr Val Thr Asp His Pro Arg 20 25 30Val Val Arg Gln Trp Phe Asn Asn Gly Ser Asn Ser Leu Gln Lys Val 35 40 45Glu Xaa Arg Val Thr Gly Leu Asp Ala Glu Tyr Thr Glu Arg Ala Pro 50 55 60Gly Lys Ile Gln Arg Thr Ala Val Leu Gln Leu Cys Leu Lys Asp Asp 65 70 7580 Val Leu Val Tyr His Ile Ile His Ser Pro Ser Ile Pro Gly Glu Leu 85 9095 His Asp Phe Leu Ser Arg Glu Asp Val Tyr Phe Tyr Gly Ala Ala Ile 100105 110 Lys Gly Asp Lys Gln Lys Leu Glu Pro Tyr Asn Leu Asp Leu Lys Ser115 120 125 Ile Ala Asp Leu Gln Thr Arg Ile Lys Ile Pro Val Glu Asp CysAsp 130 135 140 Lys Gln Thr Pro Ser Leu Phe Asp Val Ala Asn Phe Val LeuGly Thr 145 150 155 160 Asn Leu Gln Lys Gly Asp Glu Pro Val Ala Leu ArgSer Ser Gly Trp 165 170 175 Glu Asn Tyr Pro Leu Thr Tyr Glu Arg Ile LysXaa Ala Ala Leu Asp 180 185 190 23 878 DNA Zea mays 23 gcacacgaaggcatggcgac ggagatccag ggctactacg atgacggcac cgccgtggtg 60 tcgttcgacgtgcacaacat cgacaccacg ctgacgaact tgggcagcgt ggtggagtgg 120 tggctgggtgagacctatcg cctccaccac cgcggccaca tcgccggcct cgacgtggag 180 tggcgccccgctcgcgtgcc gggccccgtc cccgtcgccg tgctgcagat ctgcgtcgac 240 caccgctgcctcgtattcca gatcctccaa gccgactaca tccccgacgc cctgtccagg 300 ttcctcgccgaccgccggtt caccttcgtg ggggtcggga tcagcggcga cgtcgcaaag 360 ctgcgggccgggtacaggct gggggtggcg agcgccgtgg acctgcgcgt tctcgctgcc 420 gacacgctggaggtgcccga gctgctccgc gcggggcttc agacgctggt gtgggaggtg 480 atgggcgtgcagatggtgaa gccgcaccac gtgcgcgtca gcgcctggga cacgcccacg 540 ctgtcggaagaccagctcaa gtacgcctgc gccgacgctt tcgcctcgtt cgaggtcggc 600 cggaggctctacgaaggcga ctactagggt ctagggttac ggtatgctgt ctggtttagc 660 aatgccatgcgtgtcaggcc ggcgtatcag tattagcagt cgtcgatggt cttcagtttg 720 cgttgcagttgtgtcctcta atttctctgc ctaataagtt gtactagcta gtatcacgcg 780 cgtgatctcttttgtgtgcg ccaccactcg tcatgcatat ggcatttcga cttcaaaatt 840 tgaatgctatcttgaagccc aaaaaaaaaa aaaaaaaa 878 24 204 PRT Zea mays 24 Met Ala ThrGlu Ile Gln Gly Tyr Tyr Asp Asp Gly Thr Ala Val Val 1 5 10 15 Ser PheAsp Val His Asn Ile Asp Thr Thr Leu Thr Asn Leu Gly Ser 20 25 30 Val ValGlu Trp Trp Leu Gly Glu Thr Tyr Arg Leu His His Arg Gly 35 40 45 His IleAla Gly Leu Asp Val Glu Trp Arg Pro Ala Arg Val Pro Gly 50 55 60 Pro ValPro Val Ala Val Leu Gln Ile Cys Val Asp His Arg Cys Leu 65 70 75 80 ValPhe Gln Ile Leu Gln Ala Asp Tyr Ile Pro Asp Ala Leu Ser Arg 85 90 95 PheLeu Ala Asp Arg Arg Phe Thr Phe Val Gly Val Gly Ile Ser Gly 100 105 110Asp Val Ala Lys Leu Arg Ala Gly Tyr Arg Leu Gly Val Ala Ser Ala 115 120125 Val Asp Leu Arg Val Leu Ala Ala Asp Thr Leu Glu Val Pro Glu Leu 130135 140 Leu Arg Ala Gly Leu Gln Thr Leu Val Trp Glu Val Met Gly Val Gln145 150 155 160 Met Val Lys Pro His His Val Arg Val Ser Ala Trp Asp ThrPro Thr 165 170 175 Leu Ser Glu Asp Gln Leu Lys Tyr Ala Cys Ala Asp AlaPhe Ala Ser 180 185 190 Phe Glu Val Gly Arg Arg Leu Tyr Glu Gly Asp Tyr195 200 25 2189 DNA Zea mays 25 gcacgaggcg accggcgcct agcgttctgtggccgccgcc cttctgccgt ccgaccggac 60 caggtcgccg acgtcgcccc tatcccgcacgcgtcatcgc agcgcctccg agtctccgcc 120 aagatctgcc tcggccacgg cgcggcgccgctggagctgc agccgtccag ccgactgcct 180 tccgccgacc accgaccacc gaccactggccgagccgccg cagctagctg cgctttgaag 240 catgggtcgt tttgaacaag ttactccagaaggccctgaa actaatcagc atgatgagca 300 gcgctccata cgtttacatg cattttctgatctatcacac gtccctgcag ccacttttat 360 ttatctctta aaggactgct atggatatggtacgaacaaa gcaacctcaa agtttaagat 420 cctcatgcag ctggtaaaag tggcattgcataatggtcca cagccaggcc cgttcacata 480 tgttgtccag tgtatgtata ttgtacctctgctggggaaa acttattctg aagggttcag 540 ccatatgttg acatcctctt taaagcacttgaaatctgtg gaatcagcac agaaagattt 600 cttggaggca aagcaccttg ccgcacaacttgttcttgat atccttgatt ctattgtacc 660 ccatgagaac cgcatattgg tcaagcttcttgagacattt gaaattgagt tgagagacat 720 ggcccgtgct ttgtatgatt cagagttggacgatggtgat ctaatgaaag cacgtgaaca 780 tctcagacag caagttaagc gctgtatggaatcagaatcc aatgcacttg ctgtgactct 840 aataacacat ttttccatcc aatgctgtgatgagtccttc atcataaaat tgattaaaaa 900 caatcaatta gagattgcag agcagtgtgctattttcatg ggcaaggaaa tgatatcgct 960 agtcgttcaa aagtatctag atatgaaaatgctgaagagt gcaaacaaat tggtcaaaga 1020 acatgaactc acagaagagt ttccagatgttagctatttg tataaagaga gttcagtaaa 1080 gaagttggct gagaaaggat gctgggatattgcagaaact agggccaaga aggacacaaa 1140 actcctggaa tacctggtat atttagcaatggaagctggt tatatggaga aggttgacga 1200 gctttgcaag cgatactcta ttgaaggttatgttgattct ttggttccgg agaaggtttt 1260 ctgtgtatct gactacttag atctgaagaaattggatgtg gaagaaattg tctgggttga 1320 tgagatcaat gggctgctta atgcaacaagtgatattgaa gcttgtaaaa ttattggcat 1380 ggattgtgag tggagaccta atttcgagaaaaatactaaa tctagtaagg tctcaatcat 1440 acaaattgca tcagataaga tagccttcatttttgatctg attaaactgt atgaagatga 1500 cccaaaagca ttggacagtt gcttgaggcgtgttatgtgt tcatctaaga tactaaagct 1560 gggctatgac attcagtgtg atcttcatcaactaacgcga tcatacggag aattggaatg 1620 ttttcagtcc tatgaaatgg tacttgatatgcagaagctt ttcaaaggcg ttactggtgg 1680 cctctctgga ttgtcaaagg aaatattaggagctggtttg aacaagaccc ggcgaaatag 1740 caactgggag caacggccac taacccaaaatcagaaagag tacgctgctc ttgatgccgt 1800 ggtccttgtg catatattcc atgaacacatgcggaggcaa gcacagttcg gtgtctctga 1860 gggaagcaga gtcgagtgga ggtctcacgttgtttcccga gtgagttgca cgcgtacgcc 1920 cttgcgtttc tagtggtggg gttggacagcttggagtgaa attacttcta gcatctgcat 1980 gtgccgtctt ctaacatcta tgatgacagttctctggcat ggcatcatag gctcacagtt 2040 catccccaga agttagtgct gcgacttcgttgctcttgcc ttgtgtatat acagtatatt 2100 gttacctcca ccggagtttt tcctttcctgctgtatcgca ttcagtgctg agtgcattat 2160 tagatttgga accaaaaaaa aaaaaaaaa2189 26 563 PRT Zea mays 26 Met Gly Arg Phe Glu Gln Val Thr Pro Glu GlyPro Glu Thr Asn Gln 1 5 10 15 His Asp Glu Gln Arg Ser Ile Arg Leu HisAla Phe Ser Asp Leu Ser 20 25 30 His Val Pro Ala Ala Thr Phe Ile Tyr LeuLeu Lys Asp Cys Tyr Gly 35 40 45 Tyr Gly Thr Asn Lys Ala Thr Ser Lys PheLys Ile Leu Met Gln Leu 50 55 60 Val Lys Val Ala Leu His Asn Gly Pro GlnPro Gly Pro Phe Thr Tyr 65 70 75 80 Val Val Gln Cys Met Tyr Ile Val ProLeu Leu Gly Lys Thr Tyr Ser 85 90 95 Glu Gly Phe Ser His Met Leu Thr SerSer Leu Lys His Leu Lys Ser 100 105 110 Val Glu Ser Ala Gln Lys Asp PheLeu Glu Ala Lys His Leu Ala Ala 115 120 125 Gln Leu Val Leu Asp Ile LeuAsp Ser Ile Val Pro His Glu Asn Arg 130 135 140 Ile Leu Val Lys Leu LeuGlu Thr Phe Glu Ile Glu Leu Arg Asp Met 145 150 155 160 Ala Arg Ala LeuTyr Asp Ser Glu Leu Asp Asp Gly Asp Leu Met Lys 165 170 175 Ala Arg GluHis Leu Arg Gln Gln Val Lys Arg Cys Met Glu Ser Glu 180 185 190 Ser AsnAla Leu Ala Val Thr Leu Ile Thr His Phe Ser Ile Gln Cys 195 200 205 CysAsp Glu Ser Phe Ile Ile Lys Leu Ile Lys Asn Asn Gln Leu Glu 210 215 220Ile Ala Glu Gln Cys Ala Ile Phe Met Gly Lys Glu Met Ile Ser Leu 225 230235 240 Val Val Gln Lys Tyr Leu Asp Met Lys Met Leu Lys Ser Ala Asn Lys245 250 255 Leu Val Lys Glu His Glu Leu Thr Glu Glu Phe Pro Asp Val SerTyr 260 265 270 Leu Tyr Lys Glu Ser Ser Val Lys Lys Leu Ala Glu Lys GlyCys Trp 275 280 285 Asp Ile Ala Glu Thr Arg Ala Lys Lys Asp Thr Lys LeuLeu Glu Tyr 290 295 300 Leu Val Tyr Leu Ala Met Glu Ala Gly Tyr Met GluLys Val Asp Glu 305 310 315 320 Leu Cys Lys Arg Tyr Ser Ile Glu Gly TyrVal Asp Ser Leu Val Pro 325 330 335 Glu Lys Val Phe Cys Val Ser Asp TyrLeu Asp Leu Lys Lys Leu Asp 340 345 350 Val Glu Glu Ile Val Trp Val AspGlu Ile Asn Gly Leu Leu Asn Ala 355 360 365 Thr Ser Asp Ile Glu Ala CysLys Ile Ile Gly Met Asp Cys Glu Trp 370 375 380 Arg Pro Asn Phe Glu LysAsn Thr Lys Ser Ser Lys Val Ser Ile Ile 385 390 395 400 Gln Ile Ala SerAsp Lys Ile Ala Phe Ile Phe Asp Leu Ile Lys Leu 405 410 415 Tyr Glu AspAsp Pro Lys Ala Leu Asp Ser Cys Leu Arg Arg Val Met 420 425 430 Cys SerSer Lys Ile Leu Lys Leu Gly Tyr Asp Ile Gln Cys Asp Leu 435 440 445 HisGln Leu Thr Arg Ser Tyr Gly Glu Leu Glu Cys Phe Gln Ser Tyr 450 455 460Glu Met Val Leu Asp Met Gln Lys Leu Phe Lys Gly Val Thr Gly Gly 465 470475 480 Leu Ser Gly Leu Ser Lys Glu Ile Leu Gly Ala Gly Leu Asn Lys Thr485 490 495 Arg Arg Asn Ser Asn Trp Glu Gln Arg Pro Leu Thr Gln Asn GlnLys 500 505 510 Glu Tyr Ala Ala Leu Asp Ala Val Val Leu Val His Ile PheHis Glu 515 520 525 His Met Arg Arg Gln Ala Gln Phe Gly Val Ser Glu GlySer Arg Val 530 535 540 Glu Trp Arg Ser His Val Val Ser Arg Val Ser CysThr Arg Thr Pro 545 550 555 560 Leu Arg Phe 27 1765 DNA Zea mays unsure(14) n = A, C, G or T 27 ccaggccccg cggntccatt ntgctcccat ttcgcttcctttcccctacc gnatcgatgg 60 accacgcgcc agcgccccct ttcgccgtgc acctcgtcaccggcggcgga tcctcgtcgg 120 ggatcgccct cctactgcgc tccctcgccg ccgcccgtgtcgtcgctctt gacgcagagt 180 ggaagccgcg ccgncgcggt agccctgccg ctgccgaccctgcggncctg ggcgacgaca 240 caacgccggc gtccgagaca tctccagcgc cgccgaagttcccgacggtg acgctgctcc 300 aggtggcctg ccgcttcagc gatggtggtg gaggtgagggcgagcgcagc gaggtgtttg 360 tcgtcgacct gctttccgtg ccgctcgccg acctgtgggcaccgctgcga gagctgttcg 420 agcggcctga gacgctcaag ctggggttca ggtttaagcaggacctggtg tacctctcct 480 ccaccttctc cgccgccctc gggtgcgatt ccggattcgatagggtggag cctttcttgg 540 atgtcaccaa catttattac tatctcaagg gccatgataggcagaagaag cttccaaagg 600 agaccaagag tttggcaact atttgtgagg agctgcttggtatccttttg tccaaggaac 660 tccagtgtag tgattggtca tgccgcccct taagtgaagggcaaatacaa tatgctgcgt 720 cggatgccta ctacttgcta gacatatttg atttgttccaaaaaaggatc acaatggaag 780 gaaaatgttc atctacaaca gaacttactt cagacaggcattgctcatca gtggtgatag 840 aatgctcttc ttctggatat ggcatttgct cgggtagttgtttgatgtcc atagtaacca 900 agtacagtga gaagataata ttgacagaat ctgatgcaaaaccgcgtacc tccagacgaa 960 aagaaaaact gaagattcct gccaatgcca aacgcaaagataatgtggat tgcagtagtg 1020 aatggcaggg tccccctcca tgggatcctt ccattggtggggatgggtac ccaaagttct 1080 tgtgtgatgt gatgattgag ggcctagcta agcacttgcgatgtgttgga atagatgctg 1140 caattccatc ttcaaagaaa cctgaaccaa gggatctattaaatcaaaca tacaaggaag 1200 gaagaatatt attaacacgg gatgccaagc tcttgaaatatcaatattta gctggtaacc 1260 aggtcattaa tatcttccaa ttaaagattt ctgaggaccaacttatgtcg aggtgcacaa 1320 aatgcaatgg tagctttatt cagaaaccac ttaccctagaggaagctgtt gaagcctcaa 1380 aaggtttcca gattattccc acgtgcttgt tcaaccgaaatctggagttc tggaagtgca 1440 ccgattgcaa ccaactctac tgggagggga ctcagtaccacaatgcagtc caaaggttct 1500 tgtcagtgtg caacattagt gactaagcag cacgtacggccttggtactc atttgtaaaa 1560 ttgtaaggta ctgagatacc atcacctccg aaagatcgaaaataatctga tctttggcat 1620 ccactcgtgc acgatgaggc atgccatcca tttttagtggtttgctgttc gtcactgaga 1680 tgctgttgta aggaaaatag accttgactt atttgattaatggactttgc tccatgcaca 1740 tccaagagga aaggttctag atacg 1765 28 507 PRTZea mays UNSURE (7) Xaa = ANY AMINO ACID 28 Arg Pro Arg Gly Ser Ile XaaLeu Pro Phe Arg Phe Leu Ser Pro Thr 1 5 10 15 Xaa Ser Met Asp His AlaPro Ala Pro Pro Phe Ala Val His Leu Val 20 25 30 Thr Gly Gly Gly Ser SerSer Gly Ile Ala Leu Leu Leu Arg Ser Leu 35 40 45 Ala Ala Ala Arg Val ValAla Leu Asp Ala Glu Trp Lys Pro Arg Arg 50 55 60 Arg Gly Ser Pro Ala AlaAla Asp Pro Ala Xaa Leu Gly Asp Asp Thr 65 70 75 80 Thr Pro Ala Ser GluThr Ser Pro Ala Pro Pro Lys Phe Pro Thr Val 85 90 95 Thr Leu Leu Gln ValAla Cys Arg Phe Ser Asp Gly Gly Gly Gly Glu 100 105 110 Gly Glu Arg SerGlu Val Phe Val Val Asp Leu Leu Ser Val Pro Leu 115 120 125 Ala Asp LeuTrp Ala Pro Leu Arg Glu Leu Phe Glu Arg Pro Glu Thr 130 135 140 Leu LysLeu Gly Phe Arg Phe Lys Gln Asp Leu Val Tyr Leu Ser Ser 145 150 155 160Thr Phe Ser Ala Ala Leu Gly Cys Asp Ser Gly Phe Asp Arg Val Glu 165 170175 Pro Phe Leu Asp Val Thr Asn Ile Tyr Tyr Tyr Leu Lys Gly His Asp 180185 190 Arg Gln Lys Lys Leu Pro Lys Glu Thr Lys Ser Leu Ala Thr Ile Cys195 200 205 Glu Glu Leu Leu Gly Ile Leu Leu Ser Lys Glu Leu Gln Cys SerAsp 210 215 220 Trp Ser Cys Arg Pro Leu Ser Glu Gly Gln Ile Gln Tyr AlaAla Ser 225 230 235 240 Asp Ala Tyr Tyr Leu Leu Asp Ile Phe Asp Leu PheGln Lys Arg Ile 245 250 255 Thr Met Glu Gly Lys Cys Ser Ser Thr Thr GluLeu Thr Ser Asp Arg 260 265 270 His Cys Ser Ser Val Val Ile Glu Cys SerSer Ser Gly Tyr Gly Ile 275 280 285 Cys Ser Gly Ser Cys Leu Met Ser IleVal Thr Lys Tyr Ser Glu Lys 290 295 300 Ile Ile Leu Thr Glu Ser Asp AlaLys Pro Arg Thr Ser Arg Arg Lys 305 310 315 320 Glu Lys Leu Lys Ile ProAla Asn Ala Lys Arg Lys Asp Asn Val Asp 325 330 335 Cys Ser Ser Glu TrpGln Gly Pro Pro Pro Trp Asp Pro Ser Ile Gly 340 345 350 Gly Asp Gly TyrPro Lys Phe Leu Cys Asp Val Met Ile Glu Gly Leu 355 360 365 Ala Lys HisLeu Arg Cys Val Gly Ile Asp Ala Ala Ile Pro Ser Ser 370 375 380 Lys LysPro Glu Pro Arg Asp Leu Leu Asn Gln Thr Tyr Lys Glu Gly 385 390 395 400Arg Ile Leu Leu Thr Arg Asp Ala Lys Leu Leu Lys Tyr Gln Tyr Leu 405 410415 Ala Gly Asn Gln Val Ile Asn Ile Phe Gln Leu Lys Ile Ser Glu Asp 420425 430 Gln Leu Met Ser Arg Cys Thr Lys Cys Asn Gly Ser Phe Ile Gln Lys435 440 445 Pro Leu Thr Leu Glu Glu Ala Val Glu Ala Ser Lys Gly Phe GlnIle 450 455 460 Ile Pro Thr Cys Leu Phe Asn Arg Asn Leu Glu Phe Trp LysCys Thr 465 470 475 480 Asp Cys Asn Gln Leu Tyr Trp Glu Gly Thr Gln TyrHis Asn Ala Val 485 490 495 Gln Arg Phe Leu Ser Val Cys Asn Ile Ser Asp500 505 29 1576 DNA Triticum aestivum 29 ccggcgtgtt ccttccgcccccagcgacga cgcctcgcca gcccccccta atccgacgca 60 gttgccgact gttacggttctccagatggc ctgccgggga gaagacgggg gcaacgaggt 120 gttcgtcgtc gacctcctcgccgtgccgct cgccgacctg tgggcgccgc tgagggagct 180 gtttgagcgg cccgacgtgctgaagctggg gttccggttc aagcaggacc tcgtgtacct 240 ctccgccacc ttcacggctgccctcggatg cgactctgga ttcaacaggg tggagccttt 300 cttggatgtc accaacgtttatcactacct caaggggcat gacatgcaaa agagacttcc 360 aaaggagacc aagagtttggcttcaatatg tgaggaactg cttaatgtct ctttatccaa 420 ggaactccaa tgtagcgattggtcatgccg acccttgagc gaagggcaaa tacaatatgc 480 tgcatcagat gcctactacttgctatatat atttgatttg ttccatcaga aggtcagcat 540 tgaagaaaaa tgttcaccaacggctgaagc ttcagatgaa cattgctcac aaagggcaag 600 tgaatgttca tcgtcaggaaatgacatttg ctttgatggg tattcgacat ccatcatcac 660 gaagtacagc gacaggattttgttgacaga gtcagataca aaagcccgtt cctcaagacg 720 aaaagaaaag caaaagctgtcgagtgatgc caagtgcaaa gagaagtttg attacaatac 780 tgaatggatg ggtccccctccatgggatcc ttccgttggt ggagatggat acccaaagtt 840 tctgtgtgat gtgatgattgagggtctagc taagcacttg agatgtgttg gattagatgc 900 tgccactcca tcttgtaaaaaacctcaacc aagggaatta ttagatcaaa tctacaggga 960 aggaagaata ttactaacacgagatgccaa actcataaag tatgagtatt tggcgactaa 1020 tcaagtatac agagtgaaaagcctgctcaa acatgatcaa ctggctgagg tatgtgctta 1080 ttttgatatt ttccatcttcagatctctga ggaccgatta atgtcaagat gtacaaagtg 1140 caacggaagt ttcattcagaaaccattaac gctcgaggaa gccatagaag cctccaaagg 1200 ttttcaggtc atcccctcatgcctcttcaa ccgaaatatg gagttctgga agtgcactga 1260 ctgcaaccaa ctctactgggagggaactca gtaccacaac gcagttcaaa aattcatgtc 1320 agtctgcaac attagtgagtgagcattgta acatttcatt ctcaagacgc tcatgttgtt 1380 ttgtatgtga aattcaacatgtagtaggaa actgctcttt caataatgtc accatctatg 1440 ccttcggaca aatcccagttgcaattttac tttatgatgt acagtctaca gtatttcata 1500 tggtagtaac acatatgcaaatcatatggt agtaacatat atacattgat taccgacaaa 1560 aaaaaaaaaa aaaaaa 157630 446 PRT Triticum aestivum 30 Arg Arg Val Pro Ser Ala Pro Ser Asp AspAla Ser Pro Ala Pro Pro 1 5 10 15 Asn Pro Thr Gln Leu Pro Thr Val ThrVal Leu Gln Met Ala Cys Arg 20 25 30 Gly Glu Asp Gly Gly Asn Glu Val PheVal Val Asp Leu Leu Ala Val 35 40 45 Pro Leu Ala Asp Leu Trp Ala Pro LeuArg Glu Leu Phe Glu Arg Pro 50 55 60 Asp Val Leu Lys Leu Gly Phe Arg PheLys Gln Asp Leu Val Tyr Leu 65 70 75 80 Ser Ala Thr Phe Thr Ala Ala LeuGly Cys Asp Ser Gly Phe Asn Arg 85 90 95 Val Glu Pro Phe Leu Asp Val ThrAsn Val Tyr His Tyr Leu Lys Gly 100 105 110 His Asp Met Gln Lys Arg LeuPro Lys Glu Thr Lys Ser Leu Ala Ser 115 120 125 Ile Cys Glu Glu Leu LeuAsn Val Ser Leu Ser Lys Glu Leu Gln Cys 130 135 140 Ser Asp Trp Ser CysArg Pro Leu Ser Glu Gly Gln Ile Gln Tyr Ala 145 150 155 160 Ala Ser AspAla Tyr Tyr Leu Leu Tyr Ile Phe Asp Leu Phe His Gln 165 170 175 Lys ValSer Ile Glu Glu Lys Cys Ser Pro Thr Ala Glu Ala Ser Asp 180 185 190 GluHis Cys Ser Gln Arg Ala Ser Glu Cys Ser Ser Ser Gly Asn Asp 195 200 205Ile Cys Phe Asp Gly Tyr Ser Thr Ser Ile Ile Thr Lys Tyr Ser Asp 210 215220 Arg Ile Leu Leu Thr Glu Ser Asp Thr Lys Ala Arg Ser Ser Arg Arg 225230 235 240 Lys Glu Lys Gln Lys Leu Ser Ser Asp Ala Lys Cys Lys Glu LysPhe 245 250 255 Asp Tyr Asn Thr Glu Trp Met Gly Pro Pro Pro Trp Asp ProSer Val 260 265 270 Gly Gly Asp Gly Tyr Pro Lys Phe Leu Cys Asp Val MetIle Glu Gly 275 280 285 Leu Ala Lys His Leu Arg Cys Val Gly Leu Asp AlaAla Thr Pro Ser 290 295 300 Cys Lys Lys Pro Gln Pro Arg Glu Leu Leu AspGln Ile Tyr Arg Glu 305 310 315 320 Gly Arg Ile Leu Leu Thr Arg Asp AlaLys Leu Ile Lys Tyr Glu Tyr 325 330 335 Leu Ala Thr Asn Gln Val Tyr ArgVal Lys Ser Leu Leu Lys His Asp 340 345 350 Gln Leu Ala Glu Val Cys AlaTyr Phe Asp Ile Phe His Leu Gln Ile 355 360 365 Ser Glu Asp Arg Leu MetSer Arg Cys Thr Lys Cys Asn Gly Ser Phe 370 375 380 Ile Gln Lys Pro LeuThr Leu Glu Glu Ala Ile Glu Ala Ser Lys Gly 385 390 395 400 Phe Gln ValIle Pro Ser Cys Leu Phe Asn Arg Asn Met Glu Phe Trp 405 410 415 Lys CysThr Asp Cys Asn Gln Leu Tyr Trp Glu Gly Thr Gln Tyr His 420 425 430 AsnAla Val Gln Lys Phe Met Ser Val Cys Asn Ile Ser Glu 435 440 445 31 2251DNA Aquilegia vulgaris 31 gcacgaggct ctctttctct ctctccttct ctctgaagaaacttatttct ctttgatttg 60 aacatagttc ctccttcaat cagttcttca gaaaagcatggggattaaag aaagagcaaa 120 gaaagatgaa aacaatgaaa acagaacaat tatcttgcacactttctctg atatgtctcg 180 agtctctcca acagtttttg tatacctttt gaaagaaagttatgtgcgag gtactcagaa 240 ggcaacaaca aagttccgtg ttcttcagca acaagttctgcagatattac agaactctcc 300 acagccagga cctgctacat ttgttgtcca atgcttatatgtgttgccta tacttgggca 360 actgtacact gaaggctttg gtcacttaat gctatcttcttttcgccgtt tgcaaactgt 420 ctcagtagat ctatcagaag cacaaagcct cgcttcacaattagttgctg ccatcatggg 480 tggtgacgtg atctatgggg acccttttct gataaaactgcttgaggcat ttgatatcag 540 gatgacaaac atccagaaag ccatctcttg cggggaggagagtgatggta atttggacat 600 ggcaaaagca tgtattgaac cattcatatc tagattgattcaatcggagt cgtacacaac 660 agctgttacg ataatggaac atttttccat tcatcagtctgaccaatctt ttctagagaa 720 aatgatgcaa cagaaccagt tctcagcagc agaaaagtgggccaccttca tgggaaaggc 780 catggtatgt gcaacggtcc aaaagtatat agacatgaagatgcttagaa aggcttataa 840 gcttataaaa gagaatgatc ttgagcagga gttctcagaggcatatcata tgtgcaaaga 900 aagtgcttta aaaaggctag cagaaaaagc gtgctgggatgttgccgagt tgaaggctca 960 cggaaacaga aagcttcttg agtacctggt atatttagcaatggaagcgg gatactcaga 1020 gaaggttgat gagttatgcg agcgatacgc ccttacaggtttcgtgaaca tcgaagattc 1080 acaggcagtt cctccaaaga cacgctattt aaccattcatgaattggttt ctgaagatat 1140 catatgggta gataatgttg atggtttgct gaatgctacatcccttattg aggcttgcaa 1200 attgataggg gtggattgtg agtggaaacc aaattatataaaagggagca agccgaacaa 1260 ggtgtctatt atgcagattg cttctgaaaa gacagccttcattattgact tattgactct 1320 atctgtagct gaacccattg tcttagacac ctgcctcaaacgcattttgc tttctccaag 1380 cattctaaaa cttggttaca atttgcagtg cgacttgaagcagctgtctc attcttatga 1440 aaagatggag tgtttcaagc attacgaaat gttattggatattcaaaatg tgtttaaaga 1500 acgtaagggt ggtctctctg gactttctga gaaaatactgggagctggtt tgaataaaac 1560 aagacggaat agcaactggg agcaacgacc tctgagtcagaatcaaatgg agtatgcagc 1620 tcttgatgca gctgtgcttg ttcatatatt tcgacatatccgcaatcaaa ctcggtcctt 1680 gaatggcaaa gatgggcacg caaaacttga atggaagtcgaacatagttt cttacatggg 1740 caatagtttt aaaataccga agacaaagaa aaaatacaagaaaaaagtcg gacctgggat 1800 caatatacat tcatagacca tcttggacta tattccttcccacgaacttt tcagtttagt 1860 ttatcatcat caacaaaaag aaaagcgaat tctgtaagtttcagaaattg gaagccacaa 1920 tttgtgaata tactgtgtgt acatacatat acaagtctcccctggattca tttagtgtag 1980 gtagatgagc tgaaatttgc atcaaccagc agattctgttcgattggtcc gattgcagat 2040 tgccaagcat ccagagagtt cactcattgt ttggttttatattaactctg taatcatatc 2100 ttagattcat gcattgattt gttttcatga agatcattgtaccaaattac catgtaaaat 2160 ttctaaattc aaaatgccta tactaaatgt actttaactgaatgttagtg ttgctcactt 2220 gcttggccat taaaaaaaaa aaaaaaaaaa a 2251 32572 PRT Aquilegia vulgaris 32 Met Gly Ile Lys Glu Arg Ala Lys Lys AspGlu Asn Asn Glu Asn Arg 1 5 10 15 Thr Ile Ile Leu His Thr Phe Ser AspMet Ser Arg Val Ser Pro Thr 20 25 30 Val Phe Val Tyr Leu Leu Lys Glu SerTyr Val Arg Gly Thr Gln Lys 35 40 45 Ala Thr Thr Lys Phe Arg Val Leu GlnGln Gln Val Leu Gln Ile Leu 50 55 60 Gln Asn Ser Pro Gln Pro Gly Pro AlaThr Phe Val Val Gln Cys Leu 65 70 75 80 Tyr Val Leu Pro Ile Leu Gly GlnLeu Tyr Thr Glu Gly Phe Gly His 85 90 95 Leu Met Leu Ser Ser Phe Arg ArgLeu Gln Thr Val Ser Val Asp Leu 100 105 110 Ser Glu Ala Gln Ser Leu AlaSer Gln Leu Val Ala Ala Ile Met Gly 115 120 125 Gly Asp Val Ile Tyr GlyAsp Pro Phe Leu Ile Lys Leu Leu Glu Ala 130 135 140 Phe Asp Ile Arg MetThr Asn Ile Gln Lys Ala Ile Ser Cys Gly Glu 145 150 155 160 Glu Ser AspGly Asn Leu Asp Met Ala Lys Ala Cys Ile Glu Pro Phe 165 170 175 Ile SerArg Leu Ile Gln Ser Glu Ser Tyr Thr Thr Ala Val Thr Ile 180 185 190 MetGlu His Phe Ser Ile His Gln Ser Asp Gln Ser Phe Leu Glu Lys 195 200 205Met Met Gln Gln Asn Gln Phe Ser Ala Ala Glu Lys Trp Ala Thr Phe 210 215220 Met Gly Lys Ala Met Val Cys Ala Thr Val Gln Lys Tyr Ile Asp Met 225230 235 240 Lys Met Leu Arg Lys Ala Tyr Lys Leu Ile Lys Glu Asn Asp LeuGlu 245 250 255 Gln Glu Phe Ser Glu Ala Tyr His Met Cys Lys Glu Ser AlaLeu Lys 260 265 270 Arg Leu Ala Glu Lys Ala Cys Trp Asp Val Ala Glu LeuLys Ala His 275 280 285 Gly Asn Arg Lys Leu Leu Glu Tyr Leu Val Tyr LeuAla Met Glu Ala 290 295 300 Gly Tyr Ser Glu Lys Val Asp Glu Leu Cys GluArg Tyr Ala Leu Thr 305 310 315 320 Gly Phe Val Asn Ile Glu Asp Ser GlnAla Val Pro Pro Lys Thr Arg 325 330 335 Tyr Leu Thr Ile His Glu Leu ValSer Glu Asp Ile Ile Trp Val Asp 340 345 350 Asn Val Asp Gly Leu Leu AsnAla Thr Ser Leu Ile Glu Ala Cys Lys 355 360 365 Leu Ile Gly Val Asp CysGlu Trp Lys Pro Asn Tyr Ile Lys Gly Ser 370 375 380 Lys Pro Asn Lys ValSer Ile Met Gln Ile Ala Ser Glu Lys Thr Ala 385 390 395 400 Phe Ile IleAsp Leu Leu Thr Leu Ser Val Ala Glu Pro Ile Val Leu 405 410 415 Asp ThrCys Leu Lys Arg Ile Leu Leu Ser Pro Ser Ile Leu Lys Leu 420 425 430 GlyTyr Asn Leu Gln Cys Asp Leu Lys Gln Leu Ser His Ser Tyr Glu 435 440 445Lys Met Glu Cys Phe Lys His Tyr Glu Met Leu Leu Asp Ile Gln Asn 450 455460 Val Phe Lys Glu Arg Lys Gly Gly Leu Ser Gly Leu Ser Glu Lys Ile 465470 475 480 Leu Gly Ala Gly Leu Asn Lys Thr Arg Arg Asn Ser Asn Trp GluGln 485 490 495 Arg Pro Leu Ser Gln Asn Gln Met Glu Tyr Ala Ala Leu AspAla Ala 500 505 510 Val Leu Val His Ile Phe Arg His Ile Arg Asn Gln ThrArg Ser Leu 515 520 525 Asn Gly Lys Asp Gly His Ala Lys Leu Glu Trp LysSer Asn Ile Val 530 535 540 Ser Tyr Met Gly Asn Ser Phe Lys Ile Pro LysThr Lys Lys Lys Tyr 545 550 555 560 Lys Lys Lys Val Gly Pro Gly Ile AsnIle His Ser 565 570 33 2379 DNA Vitis sp. 33 ctgaggattt tgttgttgatacactaaagc ttcgcattca tgttggtcca tatttgaggg 60 aggtcttcaa agatccaacaaagaaaaagg ttatgcacgg ggcagatcgg gatatcattt 120 ggcttcaacg ggattttggcatatacatct gcaacatgtt tgataccgga caggcctcaa 180 gggtactgaa attggaaagaaatagtctgg agcaccttct gcaccactat tgtggagtca 240 ctgctaacaa agaatatcagaatggagatt ggagattacg tcctctcccc catgaaatgc 300 tcagatatgc tagggaagatacacactatt tactgcatat atatgattta atgagaaccc 360 aattgctctc aatggctgagctggagaatt ctaatgctct tttacttgag gtgtacaaac 420 gcagttttga tatttgcatgcagctttatg agaaggagct tttaactgat agttcatatc 480 tctatacata tggattgcagggggctcatt tcaatgctca gcagcttgct atagttgcag 540 gcctttttga atggcgagatgtggttgctc gtgctgagga tgaaagtact ggttatatat 600 tgcccaacaa aactcttcttgaaatcgcca aacagatgcc tgtcacaacc agcaagttac 660 gacgattgct gaaatcaaagcacccttatg ttgagcgcaa tcttggtcct gttgttagca 720 tcataaggca ttctatcctaaatgctgctg catttgaagc tgctgctcaa catctgaagg 780 agggtcacat tggaacagcatctgaagaca atacagttga taccactgga tttgaagcct 840 tgcctcctga atctcctacaagcataaggg ctgcggatgc tagagcagaa agttttgata 900 ctgacaatgt aataaatggtggcaagactg ataaactaca aacttttgtg agtgctaaag 960 aatatcatat ggagcctggaagcactatag atggacctgg cagcaaggga cgaggaggtt 1020 cttctgagcc tcctggtgaaagtaaagaag tgaaggatga aaaggatagt ttcattccag 1080 aagttgcaag agaaacccctgccagttcag gccagagtag atacacagac actcatacaa 1140 gtgtgtcgca gagtgagaaggttactgaag taacagttca gttgctgaag aagccaaacc 1200 gcgcctttgg agcactactagggaattcag cctcaaagag aaagctaaat tctgatccaa 1260 aaggaaagga agacatcaagttggagcaga taaaatcttc agtgaacctt ccattccatt 1320 cattttcagg cgggaacagggaggaactgt caaaactgga tacagaagag catactaaag 1380 tcctagaaac tcaaggttctgaagaacctc ttgctgtgcc agcctccaga aatgatttag 1440 aagagataat aatgtttgaagaaaactcag ggtctgatga atcagtaaat ggcaactcgg 1500 gagctgcaaa tgaacagttagaaggcaagg aggataatcc taaggggtct ggcttggaaa 1560 tggatgaggg aaatgagcctatgtctctca ctgacttatc ctccggtttt cagaagtgct 1620 cccagtcatt gaatgaaaccaggaaagcaa ggcgagttga aaaatctcag gagtccaatg 1680 gccttttgca ggtgaagccatttgactatg aagcagcgag gaaacaagtg agatttggtg 1740 aggatccaga ggaatcaagaggcaaagaag gtcgaggggg tctggtagat tcagtgagca 1800 agaaaagaag cttgggtaaaggtagagtcc agggagaaga tgagactgga gattacgcac 1860 aaggtagacg gcgtcaggcttttccggcaa ctgggaaccg aagtgtaact tttcgctgag 1920 ttgggtagca gttcatgttttatgctctcc tgtcccttca accaagatgt gaagagatgc 1980 attacaagtc ccagttgccaaagatgttac agagaataag aggacacaac aacataaagt 2040 ttattgtgaa ctgggctatattgtcattct aaggtgctgc tgtttggttg gcaagaatgt 2100 gatgatacac ctttcatggaaaatttaata caacattttc tgggaggcct ttactttttg 2160 aggctgctaa attttgatgaaactgtagaa tccatagaag ttggttctgt atgcggtctg 2220 ctcatgataa tgatcaaatgaaagccaact gaattctaga gatgaaaaag gatggaaagt 2280 gaggtgattt gagagggataggggttttga taatagtgtg tgttgatact taaaaaaaaa 2340 aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa aaaaaaaaa 2379 34 638 PRT Vitis sp. 34 Glu Asp Phe Val ValAsp Thr Leu Lys Leu Arg Ile His Val Gly Pro 1 5 10 15 Tyr Leu Arg GluVal Phe Lys Asp Pro Thr Lys Lys Lys Val Met His 20 25 30 Gly Ala Asp ArgAsp Ile Ile Trp Leu Gln Arg Asp Phe Gly Ile Tyr 35 40 45 Ile Cys Asn MetPhe Asp Thr Gly Gln Ala Ser Arg Val Leu Lys Leu 50 55 60 Glu Arg Asn SerLeu Glu His Leu Leu His His Tyr Cys Gly Val Thr 65 70 75 80 Ala Asn LysGlu Tyr Gln Asn Gly Asp Trp Arg Leu Arg Pro Leu Pro 85 90 95 His Glu MetLeu Arg Tyr Ala Arg Glu Asp Thr His Tyr Leu Leu His 100 105 110 Ile TyrAsp Leu Met Arg Thr Gln Leu Leu Ser Met Ala Glu Leu Glu 115 120 125 AsnSer Asn Ala Leu Leu Leu Glu Val Tyr Lys Arg Ser Phe Asp Ile 130 135 140Cys Met Gln Leu Tyr Glu Lys Glu Leu Leu Thr Asp Ser Ser Tyr Leu 145 150155 160 Tyr Thr Tyr Gly Leu Gln Gly Ala His Phe Asn Ala Gln Gln Leu Ala165 170 175 Ile Val Ala Gly Leu Phe Glu Trp Arg Asp Val Val Ala Arg AlaGlu 180 185 190 Asp Glu Ser Thr Gly Tyr Ile Leu Pro Asn Lys Thr Leu LeuGlu Ile 195 200 205 Ala Lys Gln Met Pro Val Thr Thr Ser Lys Leu Arg ArgLeu Leu Lys 210 215 220 Ser Lys His Pro Tyr Val Glu Arg Asn Leu Gly ProVal Val Ser Ile 225 230 235 240 Ile Arg His Ser Ile Leu Asn Ala Ala AlaPhe Glu Ala Ala Ala Gln 245 250 255 His Leu Lys Glu Gly His Ile Gly ThrAla Ser Glu Asp Asn Thr Val 260 265 270 Asp Thr Thr Gly Phe Glu Ala LeuPro Pro Glu Ser Pro Thr Ser Ile 275 280 285 Arg Ala Ala Asp Ala Arg AlaGlu Ser Phe Asp Thr Asp Asn Val Ile 290 295 300 Asn Gly Gly Lys Thr AspLys Leu Gln Thr Phe Val Ser Ala Lys Glu 305 310 315 320 Tyr His Met GluPro Gly Ser Thr Ile Asp Gly Pro Gly Ser Lys Gly 325 330 335 Arg Gly GlySer Ser Glu Pro Pro Gly Glu Ser Lys Glu Val Lys Asp 340 345 350 Glu LysAsp Ser Phe Ile Pro Glu Val Ala Arg Glu Thr Pro Ala Ser 355 360 365 SerGly Gln Ser Arg Tyr Thr Asp Thr His Thr Ser Val Ser Gln Ser 370 375 380Glu Lys Val Thr Glu Val Thr Val Gln Leu Leu Lys Lys Pro Asn Arg 385 390395 400 Ala Phe Gly Ala Leu Leu Gly Asn Ser Ala Ser Lys Arg Lys Leu Asn405 410 415 Ser Asp Pro Lys Gly Lys Glu Asp Ile Lys Leu Glu Gln Ile LysSer 420 425 430 Ser Val Asn Leu Pro Phe His Ser Phe Ser Gly Gly Asn ArgGlu Glu 435 440 445 Leu Ser Lys Leu Asp Thr Glu Glu His Thr Lys Val LeuGlu Thr Gln 450 455 460 Gly Ser Glu Glu Pro Leu Ala Val Pro Ala Ser ArgAsn Asp Leu Glu 465 470 475 480 Glu Ile Ile Met Phe Glu Glu Asn Ser GlySer Asp Glu Ser Val Asn 485 490 495 Gly Asn Ser Gly Ala Ala Asn Glu GlnLeu Glu Gly Lys Glu Asp Asn 500 505 510 Pro Lys Gly Ser Gly Leu Glu MetAsp Glu Gly Asn Glu Pro Met Ser 515 520 525 Leu Thr Asp Leu Ser Ser GlyPhe Gln Lys Cys Ser Gln Ser Leu Asn 530 535 540 Glu Thr Arg Lys Ala ArgArg Val Glu Lys Ser Gln Glu Ser Asn Gly 545 550 555 560 Leu Leu Gln ValLys Pro Phe Asp Tyr Glu Ala Ala Arg Lys Gln Val 565 570 575 Arg Phe GlyGlu Asp Pro Glu Glu Ser Arg Gly Lys Glu Gly Arg Gly 580 585 590 Gly LeuVal Asp Ser Val Ser Lys Lys Arg Ser Leu Gly Lys Gly Arg 595 600 605 ValGln Gly Glu Asp Glu Thr Gly Asp Tyr Ala Gln Gly Arg Arg Arg 610 615 620Gln Ala Phe Pro Ala Thr Gly Asn Arg Ser Val Thr Phe Arg 625 630 635 35868 DNA Zea mays 35 tgttagctag ctggctctcg acaagacaag gtgatcaagggacgacgact acagcgcgcg 60 caagccgtcc gtgtcctctc ctccctgccg gcgccggcgcaatgacgttc gcgtacgaca 120 cggacgtcgt catggacgac ggcaccatca tcaaaactaccgtcaccaac tccggcgacg 180 ccaccaagct cttcctccgc gaggtgcgcc agaccagaaagcccctgatc gtgggtctgg 240 acaccgagtg gcgcgtcatt cgccgacagg gccggcgcccgcgcaaccgg atggccgtgc 300 tgcagctctg cgtgggccac cgctgtctgg tcttccagatagtcgcggcc gactatgtcc 360 cggccgcgct gaaagccttc ctcgccagcc cgcagcaccgcttcgtcggc gtcgtggtcg 420 acgtcgacgt agagcgcttg cgctgcgact gcaacattgtggtcaataac actgtagacc 480 tgaggtatgc cgcggccgac gtgctcggcc ggccgcacctcaggacggcg gggctcaaga 540 tcctcgcccg cgaggtgatg ggagtggaaa tagagaagccgaagcacctg acctgtagcg 600 agtgggacag acctctgtcg caggcgcagg tccgctacgctgccattgac gccttcgtgt 660 catacgaggt tggccggctg gtgctcacca gggagcacgcgcaagatgcg gccttcaccg 720 gtgcaatgac gatattgccg tcgcagttgc cgtaaatgtgcatagtttta cttagttagt 780 gtagggattg tttacatggt gtatctcgca catttattcattggtttaaa gaaaatttgg 840 attcttaaaa aaaaaaaaaa aaaaaaaa 868 36 250 PRTZea mays 36 Leu Ala Ser Trp Leu Ser Thr Arg Gln Gly Asp Gln Gly Thr ThrThr 1 5 10 15 Thr Ala Arg Ala Ser Arg Pro Cys Pro Leu Leu Pro Ala GlyAla Gly 20 25 30 Ala Met Thr Phe Ala Tyr Asp Thr Asp Val Val Met Asp AspGly Thr 35 40 45 Ile Ile Lys Thr Thr Val Thr Asn Ser Gly Asp Ala Thr LysLeu Phe 50 55 60 Leu Arg Glu Val Arg Gln Thr Arg Lys Pro Leu Ile Val GlyLeu Asp 65 70 75 80 Thr Glu Trp Arg Val Ile Arg Arg Gln Gly Arg Arg ProArg Asn Arg 85 90 95 Met Ala Val Leu Gln Leu Cys Val Gly His Arg Cys LeuVal Phe Gln 100 105 110 Ile Val Ala Ala Asp Tyr Val Pro Ala Ala Leu LysAla Phe Leu Ala 115 120 125 Ser Pro Gln His Arg Phe Val Gly Val Val ValAsp Val Asp Val Glu 130 135 140 Arg Leu Arg Cys Asp Cys Asn Ile Val ValAsn Asn Thr Val Asp Leu 145 150 155 160 Arg Tyr Ala Ala Ala Asp Val LeuGly Arg Pro His Leu Arg Thr Ala 165 170 175 Gly Leu Lys Ile Leu Ala ArgGlu Val Met Gly Val Glu Ile Glu Lys 180 185 190 Pro Lys His Leu Thr CysSer Glu Trp Asp Arg Pro Leu Ser Gln Ala 195 200 205 Gln Val Arg Tyr AlaAla Ile Asp Ala Phe Val Ser Tyr Glu Val Gly 210 215 220 Arg Leu Val LeuThr Arg Glu His Ala Gln Asp Ala Ala Phe Thr Gly 225 230 235 240 Ala MetThr Ile Leu Pro Ser Gln Leu Pro 245 250 37 1047 DNA Hevea brasiliensis37 gaaaaaggac gcccctgttg ggttgtctca cagttcgcag cgcgatgaca atcagcatca 60aagaccacca actcacaaac gacactcaca atctctacga cgtcaccttc ttcaccgatc 120agatccacac tctagttacc cacgctccct ccctcgtcga ccaatggctc atcgaaaccc 180aacaacaaat tcaccaaaac cctgctatcg ttggcctgga cgtcgagtgg cgccctaatt 240tcaaccgccg tatcgagaac ccaatagcta ctctacaact ctgcattggc cgtagatgtc 300tcatctatca gctcttacat tcgcctactg tcccacaatc tcttgtggaa ttccttctca 360atgggaattt tgtgtttgtg ggggttggca ttgagagtga tgttgaaaag ctggtggagg 420attatgggtt aagcgtgaga aatactgtgg atttgagggg cttggcagcg gagaagctag 480gagtgaagga gttgaagaat gctgggttga aggatttggt gaaggaagta ttggggaagg 540aaattaagaa gcccaagagg gtcacaatga gtaggtggga caatccgtgg cttactcctg 600atcaggttca gtatgcttgt cttgatgcct ttgtgtcttc tgaaattggc aggaggttga 660attctgctgc tgctggagct ggagcttcta tttgatgggg ggcttttgaa tgtgagtttt 720ggtggctctt ccaaataaag caatgattta ggattatttt gcctaaaatt ttcatgctgg 780tttatgtggt agtcttgtga acttggatac ttttcaaatt atctgtttag cttgtttttt 840agttgctgtc atggagatga tcattgcagt caatcatcct tgtaagacaa cattgttgcg 900gccaatgctc tgctgtgttt accgtgtcct tttttttata cccagtgatg attttgttga 960tggtttagaa tttgaatcct gcgtatgtca aagattgtct tggatgtctt aaggcatttg 1020tttgattata aaaaaaaaaa aaaaaaa 1047 38 216 PRT Hevea brasiliensis 38 MetThr Ile Ser Ile Lys Asp His Gln Leu Thr Asn Asp Thr His Asn 1 5 10 15Leu Tyr Asp Val Thr Phe Phe Thr Asp Gln Ile His Thr Leu Val Thr 20 25 30His Ala Pro Ser Leu Val Asp Gln Trp Leu Ile Glu Thr Gln Gln Gln 35 40 45Ile His Gln Asn Pro Ala Ile Val Gly Leu Asp Val Glu Trp Arg Pro 50 55 60Asn Phe Asn Arg Arg Ile Glu Asn Pro Ile Ala Thr Leu Gln Leu Cys 65 70 7580 Ile Gly Arg Arg Cys Leu Ile Tyr Gln Leu Leu His Ser Pro Thr Val 85 9095 Pro Gln Ser Leu Val Glu Phe Leu Leu Asn Gly Asn Phe Val Phe Val 100105 110 Gly Val Gly Ile Glu Ser Asp Val Glu Lys Leu Val Glu Asp Tyr Gly115 120 125 Leu Ser Val Arg Asn Thr Val Asp Leu Arg Gly Leu Ala Ala GluLys 130 135 140 Leu Gly Val Lys Glu Leu Lys Asn Ala Gly Leu Lys Asp LeuVal Lys 145 150 155 160 Glu Val Leu Gly Lys Glu Ile Lys Lys Pro Lys ArgVal Thr Met Ser 165 170 175 Arg Trp Asp Asn Pro Trp Leu Thr Pro Asp GlnVal Gln Tyr Ala Cys 180 185 190 Leu Asp Ala Phe Val Ser Ser Glu Ile GlyArg Arg Leu Asn Ser Ala 195 200 205 Ala Ala Gly Ala Gly Ala Ser Ile 210215 39 814 DNA Oryza sativa 39 ggcgaccgag attgaagcat actacgacgatggcagtggc acctacctgt tgtcgttcga 60 cgaggacttc ttcgacgcaa cgctcaccaagtccggcggc aaggtggagt cttggctggg 120 cgagacgtac cgcatccacc gcagctgcggccacccgctc gtcgtcggcc tcgacgtgga 180 gtggcgcccc gccgcccccg tgccgggccccgtcgccgtg ctgcaactct gcgtcgaccg 240 ccgctgcctc gtcttccaga tcctccacgccgactacgtg cccgacgcgc tgtcccgctt 300 cctcgccgat cccaggttca ccttcgtcggcgtcggggtc cgcgacgacg ccgccaggct 360 gcgggtcggg tacgggctgg aggtgccgcgcgccgtggac ctgcgcgccc tcgccgccga 420 cacgctcggg aggcccgacc tccgccgcgcggggctgcgg gcgctggtgc gggaggtgat 480 gggcgtgcag atggacaagc cgcaccacgtgcgagtcagc gcctgggaca agcgcaacct 540 ctccgaggac cagttcaagt acgcctgcgccgacgcgttc gcgtccaggg aggtcggccg 600 gaggctctac acctgcaact gcgacggcgcatgatgatgt cgtgtccttt gtggttggac 660 ggggctttta tctttttgcc atataaatttggctatcgcc tttgtgttga tcgtactgtt 720 tactgcttgg attagtggtt ggaaccttgaacttaaatga atgtcgaccg tcatgcgatt 780 cgggttaaaa aaaaaaaaaa aaaaaaaaaaaaaa 814 40 210 PRT Oryza sativa 40 Ala Thr Glu Ile Glu Ala Tyr Tyr AspAsp Gly Ser Gly Thr Tyr Leu 1 5 10 15 Leu Ser Phe Asp Glu Asp Phe PheAsp Ala Thr Leu Thr Lys Ser Gly 20 25 30 Gly Lys Val Glu Ser Trp Leu GlyGlu Thr Tyr Arg Ile His Arg Ser 35 40 45 Cys Gly His Pro Leu Val Val GlyLeu Asp Val Glu Trp Arg Pro Ala 50 55 60 Ala Pro Val Pro Gly Pro Val AlaVal Leu Gln Leu Cys Val Asp Arg 65 70 75 80 Arg Cys Leu Val Phe Gln IleLeu His Ala Asp Tyr Val Pro Asp Ala 85 90 95 Leu Ser Arg Phe Leu Ala AspPro Arg Phe Thr Phe Val Gly Val Gly 100 105 110 Val Arg Asp Asp Ala AlaArg Leu Arg Val Gly Tyr Gly Leu Glu Val 115 120 125 Pro Arg Ala Val AspLeu Arg Ala Leu Ala Ala Asp Thr Leu Gly Arg 130 135 140 Pro Asp Leu ArgArg Ala Gly Leu Arg Ala Leu Val Arg Glu Val Met 145 150 155 160 Gly ValGln Met Asp Lys Pro His His Val Arg Val Ser Ala Trp Asp 165 170 175 LysArg Asn Leu Ser Glu Asp Gln Phe Lys Tyr Ala Cys Ala Asp Ala 180 185 190Phe Ala Ser Arg Glu Val Gly Arg Arg Leu Tyr Thr Cys Asn Cys Asp 195 200205 Gly Ala 210 41 2065 DNA Glycine max 41 gcaccaggag ctgcagaagaaacaaggagg tataaagagc tagataaccc tacaatctgc 60 aatttttttg taccgaattggttccaactg tagtggtttt aaccagggat gggtttggag 120 gagaatgtag ctaagacaagcaccaccaag gacgatgcta gccaaatgtt gaccttatgc 180 acacatgctt tctatgatttaactcatgtc tccccggtgg tatttctgtt cctgctgaaa 240 aaatgttatt actatggcacctgtaaggca acagcaaaat tccgagccct tcaacatcaa 300 gtacatcttg ttctccataatgatccaaaa cccggaccag caacttttat tgttcagtgc 360 atgtatgtct ctccattatttgaagatcac agtcaaggat ttactcatct gataatatca 420 gctcttcgcc gattcctgaaaagatcaaca atcactacag aagactcatt ggaagtgaaa 480 gacctggttg cccatctacttgtagatatt attaggggcc agatccatca tgatgaaaag 540 atagtcatga agctactcgagatttttgat gtaaaactaa caaatgttga gaaagcaatg 600 tgtcaaatta aggaaaaacacgaattaagt tgtggcacag caaacgaatt tgttgaacag 660 tatattgttg aattggtaaaatcccagttc tacatgacag ctgtcacttt aatagagcaa 720 ttctctatcc accagtatggccagtctttt ctccttgata tgatacagag taatcaattc 780 aaagcagcag agaagtgggcaacatttatg gggaaaccaa tgttatccac acttgtagag 840 gagttcattg agaggaacatgctaaagaat gcctatgaga ttataaagaa aaataatcta 900 aagcaggatt ttccagatgtatacaaaagg tgtaaagaaa gctcactaaa aaacttggca 960 gaaaaaggat gttgggatgttgctgaggca agaacaaaca atgatagaca gcttatggaa 1020 tatctggttt acttggcactggaagctggt tacatggaga aagttgatga actgtgtgat 1080 cggtactgcc tagacaggtttttggacatc aaagtacctg aaacaagtaa tctgcaaggg 1140 cgttatttac atcttgatgaattattggtt gatagcatca tttgggttga tgaagttgaa 1200 ggtttgcttg atgcaacaaggcatattaag ggttttaaag ttataggtct tgattgtgaa 1260 tggaaaccca attacgtaaaaggcagcaaa cccaacaagg tttctatcat gcaaattgct 1320 tctgaaaaga tggtttttatctttgatctg ataaagttac acaaagaagt gcctgacatt 1380 ttagatgatt gtctatcttgcattttgctg tcacctagaa ttctaaaact tggctataat 1440 ttccaatgcg atgcaaagcaacttgcttat tcatatgaag agttgagatg tttcaaaaac 1500 tatgaaatgt tgctggacatccagaatgtt tttaaagaac ctcggggtgg tttggctgga 1560 cttgcagaga aaatactgggagcaagttta aacaagacaa gacgaaacag caattgggag 1620 caacggcctt taactccaaatcaattagaa tacgctgctc tggatgctgt tgtacttgtt 1680 cacatattcc accatcttcctggtcaagga catgataaat ctgagtggaa gtcttgcatc 1740 gtgtcccaca ccgaaaacgccaagaaattc aagaaatgtg taccaaaggt tgtagacact 1800 gacatggaga ccagcaagcattgattctga attgagatcc attttcttaa ttgattacat 1860 gtgtatatgg aaaactttatatagaaaaat aagcaatttt catgagggac atgatcatta 1920 atttcattga atatatatttttttttttgt aaatctgaca gataatttaa aatttggcat 1980 tttgtacagg ctatatttactagctgcaat tacggttata taaggtgatg cccaaaatta 2040 gggcgataaa aaaaaaaaaaaaaaa 2065 42 571 PRT Glycine max 42 Met Gly Leu Glu Glu Asn Val Ala LysThr Ser Thr Thr Lys Asp Asp 1 5 10 15 Ala Ser Gln Met Leu Thr Leu CysThr His Ala Phe Tyr Asp Leu Thr 20 25 30 His Val Ser Pro Val Val Phe LeuPhe Leu Leu Lys Lys Cys Tyr Tyr 35 40 45 Tyr Gly Thr Cys Lys Ala Thr AlaLys Phe Arg Ala Leu Gln His Gln 50 55 60 Val His Leu Val Leu His Asn AspPro Lys Pro Gly Pro Ala Thr Phe 65 70 75 80 Ile Val Gln Cys Met Tyr ValSer Pro Leu Phe Glu Asp His Ser Gln 85 90 95 Gly Phe Thr His Leu Ile IleSer Ala Leu Arg Arg Phe Leu Lys Arg 100 105 110 Ser Thr Ile Thr Thr GluAsp Ser Leu Glu Val Lys Asp Leu Val Ala 115 120 125 His Leu Leu Val AspIle Ile Arg Gly Gln Ile His His Asp Glu Lys 130 135 140 Ile Val Met LysLeu Leu Glu Ile Phe Asp Val Lys Leu Thr Asn Val 145 150 155 160 Glu LysAla Met Cys Gln Ile Lys Glu Lys His Glu Leu Ser Cys Gly 165 170 175 ThrAla Asn Glu Phe Val Glu Gln Tyr Ile Val Glu Leu Val Lys Ser 180 185 190Gln Phe Tyr Met Thr Ala Val Thr Leu Ile Glu Gln Phe Ser Ile His 195 200205 Gln Tyr Gly Gln Ser Phe Leu Leu Asp Met Ile Gln Ser Asn Gln Phe 210215 220 Lys Ala Ala Glu Lys Trp Ala Thr Phe Met Gly Lys Pro Met Leu Ser225 230 235 240 Thr Leu Val Glu Glu Phe Ile Glu Arg Asn Met Leu Lys AsnAla Tyr 245 250 255 Glu Ile Ile Lys Lys Asn Asn Leu Lys Gln Asp Phe ProAsp Val Tyr 260 265 270 Lys Arg Cys Lys Glu Ser Ser Leu Lys Asn Leu AlaGlu Lys Gly Cys 275 280 285 Trp Asp Val Ala Glu Ala Arg Thr Asn Asn AspArg Gln Leu Met Glu 290 295 300 Tyr Leu Val Tyr Leu Ala Leu Glu Ala GlyTyr Met Glu Lys Val Asp 305 310 315 320 Glu Leu Cys Asp Arg Tyr Cys LeuAsp Arg Phe Leu Asp Ile Lys Val 325 330 335 Pro Glu Thr Ser Asn Leu GlnGly Arg Tyr Leu His Leu Asp Glu Leu 340 345 350 Leu Val Asp Ser Ile IleTrp Val Asp Glu Val Glu Gly Leu Leu Asp 355 360 365 Ala Thr Arg His IleLys Gly Phe Lys Val Ile Gly Leu Asp Cys Glu 370 375 380 Trp Lys Pro AsnTyr Val Lys Gly Ser Lys Pro Asn Lys Val Ser Ile 385 390 395 400 Met GlnIle Ala Ser Glu Lys Met Val Phe Ile Phe Asp Leu Ile Lys 405 410 415 LeuHis Lys Glu Val Pro Asp Ile Leu Asp Asp Cys Leu Ser Cys Ile 420 425 430Leu Leu Ser Pro Arg Ile Leu Lys Leu Gly Tyr Asn Phe Gln Cys Asp 435 440445 Ala Lys Gln Leu Ala Tyr Ser Tyr Glu Glu Leu Arg Cys Phe Lys Asn 450455 460 Tyr Glu Met Leu Leu Asp Ile Gln Asn Val Phe Lys Glu Pro Arg Gly465 470 475 480 Gly Leu Ala Gly Leu Ala Glu Lys Ile Leu Gly Ala Ser LeuAsn Lys 485 490 495 Thr Arg Arg Asn Ser Asn Trp Glu Gln Arg Pro Leu ThrPro Asn Gln 500 505 510 Leu Glu Tyr Ala Ala Leu Asp Ala Val Val Leu ValHis Ile Phe His 515 520 525 His Leu Pro Gly Gln Gly His Asp Lys Ser GluTrp Lys Ser Cys Ile 530 535 540 Val Ser His Thr Glu Asn Ala Lys Lys PheLys Lys Cys Val Pro Lys 545 550 555 560 Val Val Asp Thr Asp Met Glu ThrSer Lys His 565 570 43 432 DNA Glycine max 43 gcacgaggga tcagctaatgtcaaggtgca caaaatgcaa tggaacattt attcagaagc 60 cactgacaac tgaagaggctattgaagctg caaagggctt tcaaagaatt ccaaattgct 120 tatttaacaa gaatttagagttttggcagt gcatggactg tcaccaactt tattgggagg 180 gaacccaata ccataatgcagttcagaagt tcgttgacat ttgcaagctg agtgactaat 240 ttgaacttct ctgtataatacaaaaaagtc gttttcactt caatgtatct gttttagtgc 300 aatctattta tgtgatgtagtttatatttt gcacatagat ttggccatca ctgccccact 360 cttggttgtt cctgcgtattctactgaatg cgagaagcga ttgaaaccag atacgaatca 420 ttgaatttca at 432 44 76PRT Glycine max 44 Asp Gln Leu Met Ser Arg Cys Thr Lys Cys Asn Gly ThrPhe Ile Gln 1 5 10 15 Lys Pro Leu Thr Thr Glu Glu Ala Ile Glu Ala AlaLys Gly Phe Gln 20 25 30 Arg Ile Pro Asn Cys Leu Phe Asn Lys Asn Leu GluPhe Trp Gln Cys 35 40 45 Met Asp Cys His Gln Leu Tyr Trp Glu Gly Thr GlnTyr His Asn Ala 50 55 60 Val Gln Lys Phe Val Asp Ile Cys Lys Leu Ser Asp65 70 75 45 535 DNA Glycine max unsure (380) n = A, C, G or T 45caaacacaca gttagctctc gctccctggc tgatggatgg cggtgatcct accaaactac 60tcaaagtcca tctagtcacc tgcaccgact cggccgagtt cgcgctcctg agctcggcgc 120tgactcggac ctcggtggtg ggcctggacg cggagtggaa gcccgtccga agattgttcc 180cgagggtggc ggtgctccaa atcgcgtgcg gcgactcggc ggtgttcttg ctcgacttgc 240tgtcccttcc cctctcttcc ctgtgggccc ccttgcgcga attgctgctc tcccctgaca 300tcctcaaact cggattcgga ttcaagcaag atttggtcta cttgtcatcc actttcgcct 360cccaaggggg tttcgataan acgatttttg aattcaattg tggataaggt gaaccatatt 420tggntatcaa gagtgtctac aatcatctac aagcataata agaaacntgt tcccaagcaa 480agtaangagt ttgtcaacca aatgtgcaaa antantgggg gtttcacccc caagg 535 46 102PRT Glycine max 46 Met Asp Gly Gly Asp Pro Thr Lys Leu Leu Lys Val HisLeu Val Thr 1 5 10 15 Cys Thr Asp Ser Ala Glu Phe Ala Leu Leu Ser SerAla Leu Thr Arg 20 25 30 Thr Ser Val Val Gly Leu Asp Ala Glu Trp Lys ProVal Arg Arg Leu 35 40 45 Phe Pro Arg Val Ala Val Leu Gln Ile Ala Cys GlyAsp Ser Ala Val 50 55 60 Phe Leu Leu Asp Leu Leu Ser Leu Pro Leu Ser SerLeu Trp Ala Pro 65 70 75 80 Leu Arg Glu Leu Leu Leu Ser Pro Asp Ile LeuLys Leu Gly Phe Gly 85 90 95 Phe Lys Gln Asp Leu Val 100 47 627 DNAHelianthus sp. unsure (555) n = A, C, G or T 47 gcacgagcac ctggtgtcctccaccgattc tcccgagttc ggtcggttaa aatgggcagt 60 aagtcattcc tccatcatcggactggacgc cgaatggaag cccgtccgag ttcaccaggc 120 cacttttcct cccgttttgcttctccagat ggcctgccga ctactcaacc aagaagactc 180 cccccttgtt gttttcctactcgacctttc acagcttcct ctgcccgata tacaccacct 240 actcactcac gtattcctctctcctaatat tcttaagcta gggtttcgat ttaaacagga 300 cctgctttac ctctcctctacttttcgttc ccattctggt ggtggtggtg gtttcaatag 360 ggtggagccg tatttggatattgcaagcat atacagtagt catctacacc accacaagca 420 aaccagaaaa aagacaaacagcctttcatc catatgccag gaacttctag gcatctctct 480 ttcaaaggaa cttcaatgcagcgattggtc tctacgtcct cttacacaac accaaattac 540 ttacgccgct ttagncgctctttgtttgat tcacattttt catgtttttc agcaaagact 600 tctcnnnnnn nnnaccattcaaagcct 627 48 206 PRT Helianthus sp. UNSURE (183) Xaa = ANY AMINO ACID48 His Leu Val Ser Ser Thr Asp Ser Pro Glu Phe Gly Arg Leu Lys Trp 1 510 15 Ala Val Ser His Ser Ser Ile Ile Gly Leu Asp Ala Glu Trp Lys Pro 2025 30 Val Arg Val His Gln Ala Thr Phe Pro Pro Val Leu Leu Leu Gln Met 3540 45 Ala Cys Arg Leu Leu Asn Gln Glu Asp Ser Pro Leu Val Val Phe Leu 5055 60 Leu Asp Leu Ser Gln Leu Pro Leu Pro Asp Ile His His Leu Leu Thr 6570 75 80 His Val Phe Leu Ser Pro Asn Ile Leu Lys Leu Gly Phe Arg Phe Lys85 90 95 Gln Asp Leu Leu Tyr Leu Ser Ser Thr Phe Arg Ser His Ser Gly Gly100 105 110 Gly Gly Gly Phe Asn Arg Val Glu Pro Tyr Leu Asp Ile Ala SerIle 115 120 125 Tyr Ser Ser His Leu His His His Lys Gln Thr Arg Lys LysThr Asn 130 135 140 Ser Leu Ser Ser Ile Cys Gln Glu Leu Leu Gly Ile SerLeu Ser Lys 145 150 155 160 Glu Leu Gln Cys Ser Asp Trp Ser Leu Arg ProLeu Thr Gln His Gln 165 170 175 Ile Thr Tyr Ala Ala Leu Xaa Ala Leu CysLeu Ile His Ile Phe His 180 185 190 Val Phe Gln Gln Arg Leu Leu Xaa XaaXaa Thr Ile Gln Ser 195 200 205 49 643 PRT Mus musculus 49 Met Glu ThrThr Ser Leu Gln Arg Lys Phe Pro Glu Trp Met Ser Met 1 5 10 15 Gln SerGln Arg Cys Ala Thr Glu Glu Lys Ala Cys Val Gln Lys Asn 20 25 30 Val LeuGlu Asp Asn Leu Pro Phe Leu Glu Phe Pro Gly Ser Ile Val 35 40 45 Tyr SerTyr Glu Ala Ser Asp Cys Ser Phe Leu Ser Glu Asp Ile Ser 50 55 60 Met ArgLeu Ser Asp Gly Asp Val Val Gly Phe Asp Met Glu Trp Pro 65 70 75 80 ProIle Tyr Lys Pro Gly Lys Arg Ser Arg Val Ala Val Ile Gln Leu 85 90 95 CysVal Ser Glu Asn Lys Cys Tyr Leu Phe His Ile Ser Ser Met Ser 100 105 110Val Phe Pro Gln Gly Leu Lys Met Leu Leu Glu Asn Lys Ser Ile Lys 115 120125 Lys Ala Gly Val Gly Ile Glu Gly Asp Gln Trp Lys Leu Leu Arg Asp 130135 140 Phe Asp Val Lys Leu Glu Ser Phe Val Glu Leu Thr Asp Val Ala Asn145 150 155 160 Glu Lys Leu Lys Cys Ala Glu Thr Trp Ser Leu Asn Gly LeuVal Lys 165 170 175 His Val Leu Gly Lys Gln Leu Leu Lys Asp Lys Ser IleArg Cys Ser 180 185 190 Asn Trp Ser Asn Phe Pro Leu Thr Glu Asp Gln LysLeu Tyr Ala Ala 195 200 205 Thr Asp Ala Tyr Ala Gly Phe Ile Ile Tyr ArgLys Ile Gly Asn Phe 210 215 220 Gly Leu Ile Leu Phe Gln Val Val Ser ProIle Lys Pro Glu Glu Lys 225 230 235 240 Pro Pro Cys Asp Lys Lys Lys ProLeu Thr Leu Thr Pro Gln Glu Val 245 250 255 Met Asp Leu Ala Lys His LeuPro His Ala Phe Ser Lys Leu Glu Asn 260 265 270 Pro Arg Arg Val Ser IleLeu Leu Lys Asp Ile Ser Glu Asn Leu Cys 275 280 285 Ser Leu Arg Lys ValIle Cys Val Pro Ser Glu Trp Gly His Asp Phe 290 295 300 Arg Ser Ser PheArg Met Leu Gly Ser Leu Lys Thr Ala Leu Pro Leu 305 310 315 320 Val ProVal Ile Ala Leu Ser Ala Thr Ala Ser Ser Ser Ile Arg Glu 325 330 335 AspIle Ile Ser Cys Leu Asn Leu Lys Asp Pro Gln Ile Thr Cys Thr 340 345 350Gly Phe Asp Arg Pro Asn Leu Tyr Leu Glu Val Gly Arg Lys Thr Gly 355 360365 Asn Ile Leu Gln Asp Leu Lys Pro Phe Leu Val Arg Lys Ala Ser Ser 370375 380 Ala Trp Glu Phe Glu Gly Pro Thr Ile Ile Tyr Cys Pro Ser Arg Lys385 390 395 400 Met Thr Glu Gln Val Thr Ala Glu Leu Gly Lys Leu Asn LeuAla Cys 405 410 415 Arg Thr Tyr His Ala Gly Met Lys Ile Ser Glu Arg LysAsp Val His 420 425 430 His Arg Leu Leu Arg Asp Glu Ile Gln Cys Val ValAla Thr Val Ala 435 440 445 Phe Gly Val Gly Ile Asn Lys Ala Asp Ile ArgLys Val Ile His Asn 450 455 460 Gly Ala Pro Lys Glu Met Glu Ser Tyr TyrGln Glu Ile Gly Arg Ala 465 470 475 480 Gly Arg Asp Gly Leu Gln Ser SerCys His Leu Leu Trp Ala Pro Ala 485 490 495 Asp Phe Asn Thr Ser Arg AsnLeu Leu Ile Glu Ile His Asp Glu Lys 500 505 510 Phe Arg Leu Tyr Lys LeuLys Met Met Val Lys Met Glu Lys Tyr Leu 515 520 525 His Ser Ser Gln CysArg Arg Arg Ile Ile Leu Ser His Phe Glu Asp 530 535 540 Lys Cys Leu GlnLys Ala Ser Leu Asp Ile Met Gly Thr Glu Lys Cys 545 550 555 560 Cys AspAsn Cys Arg Pro Arg Leu Asn His Cys Leu Thr Ala Asn Asn 565 570 575 SerGlu Asp Ala Ser Gln Asp Phe Gly Pro Gln Ala Phe Gln Leu Leu 580 585 590Ser Ala Val Gly Ile Leu Gln Glu Lys Phe Gly Ile Gly Ile Pro Ile 595 600605 Leu Phe Leu Arg Gly Ser Asn Ser Gln Arg Leu Pro Asp Lys Tyr Arg 610615 620 Gly His Arg Leu Phe Gly Ala Gly Lys Glu Gln Ala Glu Ser Phe Arg625 630 635 640 Val Gly Pro 50 313 PRT Arabidopsis thaliana 50 Met SerSer Ser Asn Trp Ile Asp Asp Ala Phe Thr Glu Glu Glu Leu 1 5 10 15 LeuAla Ile Asp Ala Ile Glu Ala Ser Tyr Asn Phe Ser Arg Ser Ser 20 25 30 SerSer Ser Ser Ser Ala Ala Pro Thr Val Gln Ala Thr Thr Ser Val 35 40 45 HisGly His Glu Glu Asp Pro Asn Gln Ile Pro Asn Asn Ile Arg Arg 50 55 60 GlnLeu Pro Arg Ser Ile Thr Ser Ser Thr Ser Tyr Lys Arg Phe Pro 65 70 75 80Leu Ser Arg Cys Arg Ala Arg Asn Phe Pro Ala Met Arg Phe Gly Gly 85 90 95Arg Ile Leu Tyr Ser Lys Thr Ala Thr Glu Val Asp Lys Arg Ala Met 100 105110 Gln Leu Ile Lys Val Leu Asp Thr Lys Arg Asp Glu Ser Gly Ile Ala 115120 125 Phe Val Gly Leu Asp Ile Glu Trp Arg Pro Ser Phe Arg Lys Gly Val130 135 140 Leu Pro Gly Lys Val Ala Thr Val Gln Ile Cys Val Asp Ser AsnTyr 145 150 155 160 Cys Asp Val Met His Ile Phe His Ser Gly Ile Pro GlnSer Leu Gln 165 170 175 His Leu Ile Glu Asp Ser Thr Leu Val Lys Val GlyIle Gly Ile Asp 180 185 190 Gly Asp Ser Val Lys Leu Phe His Asp Tyr GlyVal Ser Ile Lys Asp 195 200 205 Val Glu Asp Leu Ser Asp Leu Ala Asn GlnLys Ile Gly Gly Asp Lys 210 215 220 Lys Trp Gly Leu Ala Ser Leu Thr GluThr Leu Val Cys Lys Glu Leu 225 230 235 240 Leu Lys Pro Asn Arg Ile ArgLeu Gly Asn Trp Glu Phe Tyr Pro Leu 245 250 255 Ser Lys Gln Gln Leu GlnTyr Ala Ala Thr Asp Ala Tyr Ala Ser Trp 260 265 270 His Leu Tyr Lys ValThr Thr Thr Lys Asn His Leu Leu Thr Leu Asn 275 280 285 Asp Leu Glu AlaLys Ile Ser His Arg Ser Asn Tyr Asn Thr Val Thr 290 295 300 Cys Arg LysPro Gly Gly Tyr Leu Arg 305 310 51 910 PRT Caenorhabditis elegans 51 MetGlu Glu Glu Pro Tyr Lys Arg Lys Leu Thr Lys Ala Glu Lys Lys 1 5 10 15Ala Lys Tyr Arg Thr Asp Tyr Ala Glu Pro Leu Lys Ser Arg Arg Glu 20 25 30Val Leu Lys Ala Ile Met Asn Gly Pro Glu Ser Glu Arg Glu Arg Lys 35 40 45Val Arg Ala Lys Asn Arg Glu Phe Phe Asn Glu Asp Tyr Arg Ser Gly 50 55 60Val Asn Ile Tyr Gly Met Ala Val Asp Met Met Lys Ala Met Pro Asp 65 70 7580 Arg Gly Lys Thr Ser Gly Gln Ser Leu Ala Val Trp Tyr Leu Glu Asp 85 9095 Phe Gly Val Trp Leu Lys Glu Ser Gly Gln Glu Thr Glu Leu Arg Gln 100105 110 Lys Tyr Leu Thr Gly Thr Ile Gln Ile Asn Ala Leu Asp Val Cys Thr115 120 125 Ile Gly Gln Lys Gln Leu Leu Ser Glu Ile Phe Asp Ile Thr LysGlu 130 135 140 Lys Phe Thr Glu Asp Ile Thr Gln Leu Leu Asp Ala Ala IleLys Lys 145 150 155 160 Gln Asp Phe Ser Val Ala Ala Asp Met Ala Ile GlnTyr Asn Leu Leu 165 170 175 Arg Asp His His Phe Glu His Leu Val Leu ProLeu Met Leu Ser Gly 180 185 190 Lys Asp Gln Thr Ala Tyr Lys Leu Ile SerAsn Asn Glu Arg Met Gln 195 200 205 Gln Gln Leu Val Glu Phe Phe Asp ArgMet Val Gly Ile Ser Val Val 210 215 220 Ala Val Glu Glu Met Leu Lys ProTyr Lys Glu Thr Lys Ile Met Thr 225 230 235 240 Ile Pro Met Glu Lys LeuThr Gly Lys Thr Leu Asp Lys Leu Ile Ser 245 250 255 Thr Ile Ile Asn LysAsn Thr His Glu Tyr Asn Phe Ser Arg Glu Leu 260 265 270 Ser Lys Phe AlaLys Asn His Ser Gln Asn Gly Asn Leu Lys Ala Leu 275 280 285 Lys Phe AsnIle Ser Glu Arg Tyr Glu Lys Gly Lys Ser Asp Asp Asn 290 295 300 Tyr PheGln His Met Val Glu Thr Phe Thr Lys Ala Glu Asp Val Arg 305 310 315 320Glu Pro Ile Leu Phe Tyr Leu Trp Ser Ser Asn Asp Thr Glu Lys Gln 325 330335 Ile Asp Ala Ile Cys Phe Ala Ile Tyr Leu Gly Ile Ala Ser Ser Ser 340345 350 Ser Tyr Gln Leu Pro Asn Val Met Arg Asp Phe Phe Arg Gln Pro Asp355 360 365 Ser Lys Leu Arg Glu Ala Lys Glu Leu Leu Val Arg Arg Lys ThrLeu 370 375 380 Gln Val Pro Leu Asn Gly Glu Gln Leu Phe Val Phe Glu AsnGlu Arg 385 390 395 400 Arg Thr Gln Ile His Met Val Lys Thr Glu Ser GluMet Asn Tyr Leu 405 410 415 Cys Ser Glu Ile Lys Ser Leu Ser Asp Glu ProAla Pro Val Tyr Val 420 425 430 Gly Phe Asp Ser Glu Trp Lys Pro Ser AsnLeu Thr Ala Val His Asp 435 440 445 Ser Lys Ile Ala Ile Ile Gln Leu PhePhe Lys Asn Cys Val Trp Leu 450 455 460 Val Asp Cys Val Glu Leu Glu LysAla Asn Met Ala Asp Asp Trp Trp 465 470 475 480 Gln Lys Phe Ala Ser ArgLeu Phe Gly Asp Ser Pro Val Lys Val Val 485 490 495 Gly Phe Asp Met ArgAsn Asp Leu Asp Ala Met Ala Thr Ile Pro Ala 500 505 510 Leu Lys Ser SerMet Lys Ile Glu Asp Thr Lys Asn Ala Phe Asp Leu 515 520 525 Lys Arg LeuAla Glu Asn Val Cys Asp Ile Asp Met Glu Ile Leu Glu 530 535 540 Leu ProLys Lys Thr Phe Lys Leu Ala Asp Leu Thr His Tyr Leu Leu 545 550 555 560Gly Leu Glu Leu Asp Lys Thr Glu Gln Cys Ser Asn Trp Gln Cys Arg 565 570575 Pro Leu Arg Lys Lys Gln Ile Val Tyr Ala Ala Leu Asp Ala Val Val 580585 590 Val Val Glu Thr Phe Lys Lys Ile Leu Ser Ile Val Glu Glu Lys Asn595 600 605 Lys Asp Ala Asp Ile Glu Lys Ile Val Arg Glu Ser Asn Val MetAla 610 615 620 Pro Lys Lys Asp Lys Gly His Lys Ser Tyr Arg Lys Leu LysThr Ile 625 630 635 640 Pro Trp Leu Glu Leu Tyr Asp Ile Leu Arg Ser HisArg Asn Pro Thr 645 650 655 Arg Ser Pro Gln Arg Pro His Asp Ile Lys ValIle Val Asp Thr Met 660 665 670 Leu Ile Gly Phe Gly Lys Asn Leu Arg ArgVal Gly Ile Asp Val Ile 675 680 685 Leu Pro Lys Asp Val Ser Asp Phe ArgLys Tyr Leu Lys Glu Ile Glu 690 695 700 Arg Val Gly Gly Glu His Leu ArgHis Ile Ile Thr Val Pro Ser Lys 705 710 715 720 Ser Tyr Glu Ala Leu LysMet Asp Tyr Asp Asn Tyr Thr Ile Ala Ile 725 730 735 Pro Glu Leu Asn AsnMet Ser Pro Val Asp Gln Leu Ile Glu Phe Phe 740 745 750 Asp Leu Phe AsnVal Asp Ile Arg Pro Glu Asp Val Tyr Pro Arg Cys 755 760 765 Thr Glu CysAsn Ser Arg Leu Gln Ile Lys Phe Pro Gly Pro Val Leu 770 775 780 His PheLeu His Gln Tyr Cys Val Ile His Val Gln Asn Val Tyr Arg 785 790 795 800Ala Asp Met Ser Glu Phe Pro Leu Glu Glu Trp Trp Asn Arg Met Leu 805 810815 His Ile Asn Pro Asp Asp Tyr Asp Gly Val Lys Val Glu Met Ser Arg 820825 830 Pro Ser Pro Thr Ser Lys Trp Ile Val Ala Thr Val Pro Thr Gly Cys835 840 845 Leu His Ile Thr Arg Gln Thr Ala Leu His Thr Asn Leu Pro AspGly 850 855 860 Ile Glu Val Arg Ile His Lys Val Pro Asp Asp Glu Phe LysArg Arg 865 870 875 880 Asn Leu Ser Phe Tyr Val Cys Gly Glu Cys Gly ThrVal Ala Cys Asp 885 890 895 Gly Arg Gly Asn Gln Ala Ser Glu Ser Thr SerGln Glu Cys 900 905 910 52 217 PRT Arabidopsis thaliana 52 Met Lys ArgGly Ile Lys His Leu Cys Phe Asn Gly Phe Thr Gly Tyr 1 5 10 15 Ser SerLeu His His His Tyr His Glu His His Val Asp Phe Phe Gly 20 25 30 Glu ArgLeu Ile Val Thr Val Thr His Thr Pro Ser Val Ile Arg Arg 35 40 45 Trp IleHis Ser Ile Arg Phe Val Ser Arg Leu Arg Leu Ser His Pro 50 55 60 Leu ValVal Gly Leu Gly Val Gln Trp Thr Pro Arg Gly Ser Asp Pro 65 70 75 80 ProPro Asp Ile Leu Gln Leu Cys Val Gly Thr Arg Cys Leu Ile Ile 85 90 95 GlnLeu Ser His Cys Lys Tyr Val Pro Asp Val Leu Arg Ser Phe Leu 100 105 110Glu Asp Gln Thr Ile Thr Phe Val Gly Val Trp Asn Ser Gln Asp Lys 115 120125 Asp Lys Leu Glu Arg Phe His His Gln Leu Asp Ile Trp Arg Leu Val 130135 140 His Ile Arg His Tyr Leu His Pro Leu Leu Leu Ser Ser Ser Phe Glu145 150 155 160 Thr Ile Val Lys Val Tyr Leu Gly His Glu Gly Val Thr LysAsp Lys 165 170 175 Glu Leu Cys Met Ser Asn Trp Gly Ala Arg Ser Leu SerHis Asp Gln 180 185 190 Ile Val Gln Ala Ser His Asp Val Tyr Val Cys CysLys Leu Gly Val 195 200 205 Lys Glu Arg Leu Trp Lys Met Gly Ala 210 21553 155 PRT Pyrococcus abyssi 53 Met Lys Phe Ile Ala Asp Met Met Leu GlyArg Leu Ala Arg Trp Leu 1 5 10 15 Arg Leu Tyr Gly Tyr Asp Thr Lys TyrGly Ile Lys Asp Asp Asp Glu 20 25 30 Ile Ile Glu Thr Ala Lys Lys Glu GlyArg Ile Ile Leu Ser Arg Asp 35 40 45 Leu Glu Leu Val Glu Arg Ala Lys LysLeu Gly Ile Lys Ala Ile Phe 50 55 60 Ile Glu Ser Asn Ser Ile Glu Gly GlnIle Ala Gln Leu Met Arg Leu 65 70 75 80 Gly Ile Glu Phe Asn Glu Leu PhePro Glu Gly Ala Arg Cys Pro Lys 85 90 95 Cys Asn Gly Ile Ile Val Arg ValSer Lys Glu Glu Val Lys Gly Lys 100 105 110 Val Pro Glu Lys Val Tyr GluSer Tyr Asp Glu Phe Tyr Val Cys Thr 115 120 125 Asn Cys Gly Gln Ile TyrTrp Pro Gly Arg Gln Trp Glu Glu Met Val 130 135 140 Lys Met Asp Lys LysPhe Arg Thr Asn Lys Pro 145 150 155 54 505 PRT Arabidopsis thaliana 54Met Glu Thr Asn Leu Lys Ile Tyr Leu Val Ser Ser Thr Asp Ser Ser 1 5 1015 Glu Phe Thr His Leu Lys Trp Ser Phe Thr Arg Ser Thr Ile Ile Ala 20 2530 Leu Asp Ala Glu Trp Lys Pro Gln His Ser Asn Thr Ser Ser Phe Pro 35 4045 Thr Val Thr Leu Leu Gln Val Ala Cys Arg Leu Ser His Ala Thr Asp 50 5560 Val Ser Asp Val Phe Leu Ile Asp Leu Ser Ser Ile His Leu Pro Ser 65 7075 80 Val Trp Glu Leu Leu Asn Asp Met Phe Val Ser Pro Asp Val Leu Lys 8590 95 Leu Gly Phe Arg Phe Lys Gln Asp Leu Val Tyr Leu Ser Ser Thr Phe100 105 110 Thr Gln His Gly Cys Glu Gly Gly Phe Gln Glu Val Lys Gln TyrLeu 115 120 125 Asp Ile Thr Ser Ile Tyr Asn Tyr Leu Gln His Lys Arg PheGly Arg 130 135 140 Lys Ala Pro Lys Asp Ile Lys Ser Leu Ala Ala Ile CysLys Glu Met 145 150 155 160 Leu Asp Ile Ser Leu Ser Lys Glu Leu Gln CysSer Asp Trp Ser Tyr 165 170 175 Arg Pro Leu Thr Glu Glu Gln Lys Leu TyrAla Ala Thr Asp Ala His 180 185 190 Cys Leu Leu Gln Ile Phe Asp Val PheGlu Ala His Leu Val Glu Gly 195 200 205 Ile Thr Val Gln Asp Leu Arg ValIle Asn Val Gly Leu Gln Glu Ile 210 215 220 Leu Thr Glu Ser Asp Tyr SerSer Lys Ile Val Thr Val Lys Leu Cys 225 230 235 240 Lys Ala Thr Asp ValIle Arg Ser Met Ser Glu Asn Gly Gln Asn Ile 245 250 255 Ala Asn Gly ValVal Pro Arg Lys Thr Thr Leu Asn Thr Met Pro Met 260 265 270 Asp Glu AsnLeu Leu Lys Ile Val Arg Lys Phe Gly Glu Arg Ile Leu 275 280 285 Leu LysGlu Ser Asp Leu Leu Pro Lys Lys Leu Lys Lys Lys Thr Arg 290 295 300 ArgArg Val Ala Ser Ser Thr Met Asn Thr Asn Lys Gln Leu Val Cys 305 310 315320 Ser Ala Asp Trp Gln Gly Pro Pro Pro Trp Asp Ser Ser Leu Gly Gly 325330 335 Asp Gly Cys Pro Lys Phe Leu Leu Asp Val Met Val Glu Gly Leu Ala340 345 350 Lys His Leu Arg Cys Val Gly Ile Asp Ala Ala Ile Pro His SerLys 355 360 365 Lys Pro Asp Ser Arg Glu Leu Leu Asp Gln Ala Phe Lys GluAsn Arg 370 375 380 Val Leu Leu Thr Arg Asp Thr Lys Leu Leu Arg His GlnAsp Leu Ala 385 390 395 400 Lys His Gln Ile Tyr Arg Val Lys Ser Leu LeuLys Asn Glu Gln Leu 405 410 415 Leu Glu Val Ile Glu Thr Phe Gln Leu LysIle Ser Gly Asn Gln Leu 420 425 430 Met Ser Arg Cys Thr Lys Cys Asn GlyLys Phe Ile Gln Lys Pro Leu 435 440 445 Ser Ile Glu Glu Ala Ile Glu AlaAla Lys Gly Phe Gln Arg Ile Pro 450 455 460 Asn Cys Leu Phe Asn Lys AsnLeu Glu Phe Trp Gln Cys Met Asn Cys 465 470 475 480 His Gln Leu Tyr TrpGlu Gly Thr Gln Tyr His Asn Ala Val Gln Lys 485 490 495 Phe Met Glu ValCys Lys Leu Ser Glu 500 505 55 582 PRT Arabidopsis thaliana 55 Met GlyLeu Asp Ser Lys Glu Ala Asp Leu Glu Val Ile Arg Asp Glu 1 5 10 15 LysSer Glu Ala Asn Thr Val Cys Leu His Ala Phe Ser Asp Leu Thr 20 25 30 TyrVal Ser Pro Val Val Phe Leu Tyr Leu Leu Lys Glu Cys Tyr Lys 35 40 45 HisGly Ser Leu Lys Ala Thr Lys Lys Phe Gln Ala Leu Gln Tyr Gln 50 55 60 ValHis Arg Val Leu Ala Asn Lys Pro Gln Pro Gly Pro Ala Thr Phe 65 70 75 80Ile Ile Asn Cys Leu Thr Leu Leu Pro Leu Phe Gly Val Tyr Gly Glu 85 90 95Gly Phe Ser His Leu Val Ile Ser Ala Leu Arg Arg Phe Phe Lys Thr 100 105110 Val Ser Glu Pro Thr Ser Glu Glu Asp Ile Cys Leu Ala Arg Lys Leu 115120 125 Ala Ala Gln Phe Phe Leu Ala Thr Val Gly Gly Ser Leu Thr Tyr Asp130 135 140 Glu Lys Val Met Val His Thr Leu Arg Val Phe Asp Val Arg LeuThr 145 150 155 160 Ser Ile Asp Glu Ala Leu Ser Ile Ser Glu Val Trp GlnArg Tyr Gly 165 170 175 Phe Ala Cys Gly Asn Ala Phe Leu Glu Gln Tyr IleSer Asp Leu Ile 180 185 190 Lys Ser Lys Ser Phe Met Thr Ala Val Thr LeuLeu Glu His Phe Ser 195 200 205 Phe Arg Phe Pro Gly Glu Thr Phe Leu GlnGln Met Val Glu Asp Lys 210 215 220 Asn Phe Gln Ala Ala Glu Arg Trp AlaThr Phe Met Gly Arg Pro Ser 225 230 235 240 Leu Cys Ile Leu Val Gln GluTyr Gly Ser Arg Asn Met Leu Lys Gln 245 250 255 Ala Tyr Asn Ile Ile AsnLys Asn Tyr Leu Gln His Asp Phe Pro Glu 260 265 270 Leu Tyr His Lys CysLys Glu Ser Ala Leu Lys Val Leu Ala Glu Lys 275 280 285 Ala Cys Trp AspVal Ala Glu Ile Lys Thr Lys Gly Asp Arg Gln Leu 290 295 300 Leu Lys TyrLeu Val Tyr Leu Ala Val Glu Ala Gly Tyr Leu Glu Lys 305 310 315 320 ValAsp Glu Leu Cys Asp Arg Tyr Ser Leu Gln Gly Leu Pro Lys Ala 325 330 335Arg Glu Ala Glu Val Ala Phe Val Glu Lys Ser Phe Leu Arg Leu Asn 340 345350 Asp Leu Ala Val Glu Asp Val Val Trp Val Asp Glu Val Asn Glu Leu 355360 365 Arg Lys Ala Thr Ser Phe Leu Glu Gly Cys Arg Val Val Gly Ile Asp370 375 380 Cys Glu Trp Lys Pro Asn Tyr Ile Lys Gly Ser Lys Gln Asn LysVal 385 390 395 400 Ser Ile Met Gln Ile Gly Ser Asp Thr Lys Ile Phe IleLeu Asp Leu 405 410 415 Ile Lys Leu Tyr Asn Asp Ala Ser Glu Ile Leu AspAsn Cys Leu Ser 420 425 430 His Ile Leu Gln Ser Lys Ser Thr Leu Lys LeuVal Ser Leu Thr Glu 435 440 445 Asp Tyr Pro Asp His Lys Leu Ser Ser GlyTyr Asn Phe Gln Cys Asp 450 455 460 Ile Lys Gln Leu Ala Leu Ser Tyr GlyAsp Leu Lys Cys Phe Glu Arg 465 470 475 480 Tyr Asp Met Leu Leu Asp IleGln Asn Val Phe Asn Glu Pro Phe Gly 485 490 495 Gly Leu Ala Gly Leu ThrLys Lys Ile Leu Gly Val Ser Leu Asn Lys 500 505 510 Thr Arg Arg Asn SerAsp Trp Glu Gln Arg Pro Leu Ser Gln Asn Gln 515 520 525 Leu Glu Tyr AlaAla Leu Asp Ala Ala Val Leu Ile His Ile Phe Arg 530 535 540 His Val ArgAsp His Pro Pro His Asp Ser Ser Ser Glu Thr Thr Gln 545 550 555 560 TrpLys Ser His Ile Val Ser Thr Ser Tyr Lys Ser Pro Tyr Leu Ser 565 570 575Ser Asp Asn Ser Arg Arg 580 56 2376 PRT Arabidopsis thaliana 56 Met GluSer Pro Gly Arg Lys Val Leu Tyr Glu Ile Arg His His Ala 1 5 10 15 SerLeu Pro Tyr Val Pro Arg Tyr Pro Pro Leu Pro Gln Ala Asp Gly 20 25 30 ThrAsn Ser Lys Gly Gly Leu Arg Ser Leu Val Ser Ile Lys Gly Val 35 40 45 SerGln Leu Lys Glu Lys Trp Ser Glu Tyr Trp Asn Pro Lys Lys Thr 50 55 60 AsnLys Pro Val Ser Leu Phe Ile Ser Pro Arg Gly Glu Leu Val Ala 65 70 75 80Val Thr Ser Gly Asn His Val Thr Ile Leu Arg Lys Asp Asp Asp Tyr 85 90 95Arg Lys Pro Cys Gly Asn Phe Thr Ser Ser Ile Ser Gly Ser Phe Thr 100 105110 Ser Gly Val Trp Ser Glu Lys His Asp Val Leu Gly Leu Val Asp Asp 115120 125 Ser Glu Thr Leu Phe Phe Ile Arg Ala Asn Gly Glu Glu Ile Ser Gln130 135 140 Val Thr Lys Arg Asn Leu Lys Val Ser Ala Pro Val Leu Gly LeuMet 145 150 155 160 Glu Asp Asp Ser Asp Leu Gln Pro Ser Cys Leu Cys SerPhe Ser Ile 165 170 175 Leu Thr Ser Asp Gly Arg Ile His His Val Glu IleSer Arg Glu Pro 180 185 190 Ser Ala Ser Ala Phe Ser Lys His Ala Ser AsnSer Val Ser Lys Gln 195 200 205 Phe Pro Asn His Val Phe Cys Phe Asp TyrHis Pro Asp Leu Ser Phe 210 215 220 Leu Leu Ile Val Gly Ser Val Ala GlyIle Ser Ser Ser Gly Ser Ser 225 230 235 240 Gly Ser Ser Cys Ile Ser LeuTrp Arg Lys Cys Gln Asn Leu Gly Leu 245 250 255 Glu Leu Leu Ser Thr ThrLys Phe Asp Gly Val Tyr Cys Glu Asn Lys 260 265 270 Asp Asp Gln Leu AlaTyr Pro Lys Thr Leu Ile Ser Pro Gln Gly Ser 275 280 285 His Val Ala SerLeu Asp Ser Asn Gly Cys Val His Ile Phe Gln Leu 290 295 300 Asp Lys AlaArg Leu Thr Leu Ser Cys Cys Pro Ser Glu Asp Ser Ser 305 310 315 320 AspSer Leu Lys Pro Asp Lys Ser Leu Gln Ser Trp Lys Glu Ser Leu 325 330 335Arg Asn Val Val Asp Phe Thr Trp Trp Ser Asp His Ala Leu Ala Ile 340 345350 Leu Lys Arg Ser Gly Asn Ile Ser Ile Phe Asp Ile Ser Arg Cys Val 355360 365 Ile Val Gln Glu Asp Ala Thr Ile Tyr Ser Met Pro Val Val Glu Arg370 375 380 Val Gln Lys Tyr Glu Gly His Ile Phe Leu Leu Glu Ser Ser ThrGln 385 390 395 400 Glu Ala Lys Ser Ala Leu Ala Asn Val Asp Arg Asp AlaSer Glu Phe 405 410 415 His His Thr Ser Glu His Ser Met Leu Trp Arg LeuIle Ser Phe Thr 420 425 430 Glu Lys Thr Ile Pro Glu Met Tyr Lys Ile LeuVal Glu Lys Cys Gln 435 440 445 Tyr Gln Glu Ala Leu Asp Phe Ser Asp SerHis Gly Leu Asp Arg Asp 450 455 460 Glu Val Phe Lys Ser Arg Trp Leu LysSer Glu Lys Gly Val Ser Asp 465 470 475 480 Val Ser Thr Ile Leu Ser LysIle Lys Asp Lys Ala Phe Val Leu Ser 485 490 495 Glu Cys Leu Asp Arg IleGly Pro Thr Glu Asp Ser Met Lys Ala Leu 500 505 510 Leu Ala His Gly LeuTyr Leu Thr Asn His Tyr Val Phe Ala Lys Ser 515 520 525 Glu Asp Gln GluSer Gln Gln Leu Trp Glu Phe Arg Leu Ala Arg Leu 530 535 540 Arg Leu LeuGln Phe Ser Glu Arg Leu Asp Thr Tyr Leu Gly Ile Ser 545 550 555 560 MetGly Arg Tyr Ser Val Gln Asp Tyr Arg Lys Phe Arg Ser Asn Pro 565 570 575Ile Asn Gln Ala Ala Ile Ser Leu Ala Glu Ser Gly Arg Ile Gly Ala 580 585590 Leu Asn Leu Leu Phe Lys Arg His Pro Tyr Ser Leu Val Ser Phe Met 595600 605 Leu Gln Ile Leu Ala Ala Ile Pro Glu Thr Val Pro Val Glu Thr Tyr610 615 620 Ala His Leu Leu Pro Gly Lys Ser Pro Pro Thr Ser Met Ala ValArg 625 630 635 640 Glu Glu Asp Trp Val Glu Cys Glu Lys Met Val Lys PheIle Asn Asn 645 650 655 Leu Pro Glu Asn Gly Lys Asn Asp Ser Leu Ile GlnThr Glu Pro Ile 660 665 670 Val Arg Arg Cys Leu Gly Tyr Asn Trp Pro SerSer Glu Glu Leu Ala 675 680 685 Ala Trp Tyr Lys Ser Arg Ala Arg Asp IleAsp Ser Thr Thr Gly Leu 690 695 700 Leu Asp Asn Cys Ile Cys Leu Ile AspIle Ala Cys Arg Lys Gly Ile 705 710 715 720 Ser Glu Leu Glu Gln Phe HisGlu Asp Leu Ser Tyr Leu His Gln Ile 725 730 735 Ile Tyr Ser Asp Glu IleGly Gly Glu Ile Cys Phe Ser Leu Ser Leu 740 745 750 Ala Gly Trp Glu HisLeu Ser Asp Tyr Glu Lys Phe Lys Ile Met Leu 755 760 765 Glu Gly Val LysAla Asp Thr Val Val Arg Arg Leu His Glu Lys Ala 770 775 780 Ile Pro PheMet Gln Lys Arg Phe Leu Gly Thr Asn Asn Gln Asn Val 785 790 795 800 GluSer Phe Leu Val Lys Trp Leu Lys Glu Met Ala Ala Lys Ser Asp 805 810 815Met Asp Leu Cys Ser Lys Val Ile Asp Glu Gly Cys Ile Asp Leu Tyr 820 825830 Thr Val Cys Phe Phe Lys Asp Asp Val Glu Ala Val Asp Cys Ala Leu 835840 845 Gln Cys Leu Tyr Leu Cys Lys Val Thr Asp Lys Trp Asn Val Met Ala850 855 860 Thr Met Leu Ser Lys Leu Pro Lys Ile Asn Asp Lys Ala Gly GluAsp 865 870 875 880 Ile Gln Arg Arg Leu Lys Arg Ala Glu Gly His Ile GluAla Gly Arg 885 890 895 Leu Leu Glu Phe Tyr Gln Val Pro Lys Pro Ile AsnTyr Phe Leu Glu 900 905 910 Val His Leu Asp Glu Lys Gly Val Lys Gln IleLeu Arg Leu Met Leu 915 920 925 Ser Lys Phe Val Arg Arg Gln Pro Gly ArgSer Asp Asn Asp Trp Ala 930 935 940 Cys Met Trp Arg Asp Leu Arg Gln LeuGln Glu Lys Ala Phe Tyr Phe 945 950 955 960 Leu Asp Leu Glu Phe Val LeuThr Glu Phe Cys Arg Gly Leu Leu Lys 965 970 975 Ala Gly Lys Phe Ser LeuAla Arg Asn Tyr Leu Lys Gly Thr Gly Ser 980 985 990 Val Ala Leu Pro SerGlu Lys Ala Glu Ser Leu Val Ile Asn Ala Ala 995 1000 1005 Lys Glu TyrPhe Phe Ser Ala Pro Ser Leu Ala Ser Glu Glu Ile Trp 1010 1015 1020 LysAla Arg Glu Cys Leu Asn Ile Phe Ser Ser Ser Arg Thr Val Lys 1025 10301035 1040 Ala Glu Asp Asp Ile Ile Asp Ala Val Thr Val Arg Leu Pro LysLeu 1045 1050 1055 Gly Val Ser Leu Leu Pro Val Gln Phe Lys Gln Val LysAsp Pro Met 1060 1065 1070 Glu Ile Ile Lys Met Ala Ile Thr Gly Asp ProGlu Ala Tyr Leu His 1075 1080 1085 Gly Glu Glu Leu Ile Glu Val Ala LysLeu Leu Gly Leu Asn Ser Ser 1090 1095 1100 Glu Asp Ile Ser Ser Val LysGlu Ala Ile Ala Arg Glu Ala Ala Ile 1105 1110 1115 1120 Ala Gly Asp MetGln Leu Ala Phe Asp Leu Cys Leu Val Leu Thr Lys 1125 1130 1135 Glu GlyHis Gly Pro Ile Trp Asp Leu Gly Ala Ala Ile Ala Arg Ser 1140 1145 1150Pro Ala Leu Glu His Met Asp Ile Ser Ser Arg Lys Gln Leu Leu Gly 11551160 1165 Phe Ala Leu Gly His Cys Asp Asp Glu Ser Ile Ser Glu Leu LeuHis 1170 1175 1180 Ala Trp Lys Asp Phe Asp Leu Gln Gly Gln Cys Glu ThrLeu Gly Met 1185 1190 1195 1200 Leu Ser Glu Ser Asn Ser Pro Glu Phe GlnLys Met Asp Gly Val Ser 1205 1210 1215 Cys Leu Thr Asp Phe Pro Gln MetLeu Asp Gly Leu Ser Ser Asp Gln 1220 1225 1230 Gln Leu Asp Leu Asp ArgAla Lys Asp Ser Ile Ser Cys Val Ala Lys 1235 1240 1245 Asp Met Pro ValAsp Asp Ser Val Asp Leu Glu Ser Leu Leu Lys Glu 1250 1255 1260 Asn GlyLys Leu Phe Ser Phe Ala Ala Ser His Leu Pro Trp Leu Leu 1265 1270 12751280 Lys Leu Gly Arg Asn Arg Lys Leu Asp Lys Ser Leu Val Leu Asp Ser1285 1290 1295 Ile Pro Gly Lys Gln Phe Val Ser Ile Lys Ala Thr Ala LeuIle Thr 1300 1305 1310 Ile Leu Ser Trp Leu Ala Lys Asn Gly Phe Ala ProLys Asp Glu Leu 1315 1320 1325 Ile Ala Met Ile Thr Asp Ser Ile Ile GluHis Pro Val Thr Lys Glu 1330 1335 1340 Glu Asp Val Ile Gly Cys Ser PheLeu Leu Asn Leu Val Asp Ala Ser 1345 1350 1355 1360 Asn Ala Val Glu ValIle Glu Lys Gln Leu Arg Ile Arg Gly Asn Tyr 1365 1370 1375 Gln Glu IleArg Ser Ile Met Ser Leu Gly Met Ile Tyr Ser Leu Leu 1380 1385 1390 HisAsp Ser Gly Val Glu Cys Thr Ala Pro Ile Gln Arg Arg Glu Leu 1395 14001405 Leu Gln Lys Asn Phe Glu Arg Lys Gln Thr Glu Ser Leu Ala Asp Asp1410 1415 1420 Met Ser Lys Ile Asp Lys Leu Gln Ser Thr Phe Trp Lys GluTrp Lys 1425 1430 1435 1440 His Lys Leu Glu Glu Lys Met His Asp Ala AspArg Ser Arg Met Leu 1445 1450 1455 Glu Arg Ile Ile Pro Gly Val Glu ThrGlu Arg Phe Leu Ser His Asp 1460 1465 1470 Ile Glu Tyr Ile Lys Val AlaVal Phe Ser Leu Ile Glu Ser Val Lys 1475 1480 1485 Ser Glu Lys Lys LeuIle Leu Lys Asp Val Leu Lys Leu Ala Asp Thr 1490 1495 1500 Tyr Gly LeuLys Gln Ser Glu Val Ile Leu Arg Tyr Leu Ser Ser Ile 1505 1510 1515 1520Leu Cys Ser Glu Ile Trp Thr Asn Glu Asp Ile Thr Ala Glu Ile Leu 15251530 1535 Gln Val Lys Glu Glu Ile Leu Thr Phe Ala Ser Asp Thr Ile GluThr 1540 1545 1550 Ile Ser Thr Ile Val Tyr Pro Ala Ala Ser Gly Leu AsnLys Gln Arg 1555 1560 1565 Leu Ala Tyr Ile Tyr Ser Leu Leu Ser Glu CysTyr Cys His Leu Ala 1570 1575 1580 Glu Ser Lys Glu Ala Ser Leu Leu ValGln Pro Asn Ser Ser Phe Ala 1585 1590 1595 1600 Gly Leu Ser Asn Trp TyrAsn Val Leu Lys Gln Glu Cys Ser Arg Val 1605 1610 1615 Ser Phe Ile LysAsp Leu Asp Phe Lys Asn Ile Ser Glu Leu Gly Gly 1620 1625 1630 Leu AsnPhe Asp Ser Phe Asn Asn Glu Val His Ala His Ile Asn Glu 1635 1640 1645Met Asn Leu Glu Ala Leu Ala Lys Met Val Glu Thr Leu Ser Gly Leu 16501655 1660 Ser Met Glu Asn Ser Ser Lys Gly Leu Ile Ser Cys Gln Asp ValTyr 1665 1670 1675 1680 Lys Gln Tyr Ile Met Asn Leu Leu Asp Thr Leu GluSer Arg Arg Asp 1685 1690 1695 Leu Asp Phe Gly Ser Ala Glu Ser Phe GlnGly Phe Leu Gly Gln Leu 1700 1705 1710 Glu Lys Thr Tyr Asp His Cys ArgVal Tyr Val Arg Ile Leu Glu Pro 1715 1720 1725 Leu Gln Ala Val Glu IleLeu Lys Arg His Phe Thr Leu Val Leu Pro 1730 1735 1740 Pro Asn Gly SerTyr Met His Ile Pro Asp Ser Ser Thr Trp Gln Glu 1745 1750 1755 1760 CysLeu Ile Leu Leu Ile Asn Phe Trp Ile Arg Leu Ala Asp Glu Met 1765 17701775 Gln Glu Val Lys Ser Ser Asn Pro Ser Leu Val Glu Asn Leu Thr Leu1780 1785 1790 Ser Pro Glu Cys Ile Ser Ser Cys Phe Thr Leu Leu Ile LysLeu Val 1795 1800 1805 Met Tyr Asp Ser Leu Ser Pro Ser Gln Ala Trp AlaAla Ile Leu Val 1810 1815 1820 Tyr Leu Arg Ser Gly Leu Val Gly Asp CysAla Thr Glu Ile Phe Asn 1825 1830 1835 1840 Phe Cys Arg Ala Met Val PheSer Gly Cys Gly Phe Gly Pro Ile Ser 1845 1850 1855 Asp Val Phe Ser AspMet Ser Ser Arg Tyr Pro Thr Ala Leu Gln Asp 1860 1865 1870 Leu Pro HisLeu Tyr Leu Ser Val Leu Glu Pro Ile Leu Gln Asp Leu 1875 1880 1885 ValSer Gly Ala Pro Glu Thr Gln Asn Leu Tyr Arg Leu Leu Ser Ser 1890 18951900 Leu Ser Asn Leu Glu Gly Asn Leu Glu Glu Leu Lys Arg Val Arg Leu1905 1910 1915 1920 Val Val Trp Lys Gln Leu Val Ile Phe Ser Glu Asn LeuGlu Leu Pro 1925 1930 1935 Ser Gln Val Arg Val Tyr Ser Leu Glu Leu MetGln Phe Ile Ser Gly 1940 1945 1950 Lys Asn Ile Lys Gly Ser Ser Ser GluLeu Gln Ser Asn Val Met Pro 1955 1960 1965 Trp Asp Gly Ser Ala Glu LeuLeu Ser Ser Met Gln Lys Thr Glu Ala 1970 1975 1980 Ala Leu Asn Gln AlaLeu Pro Asp Gln Ala Asp Gly Ser Ser Arg Leu 1985 1990 1995 2000 Thr AsnThr Leu Val Ala Leu Lys Ser Ser Gln Val Ala Val Ala Ala 2005 2010 2015Ile Ser Pro Gly Leu Glu Ile Ser Pro Glu Asp Leu Ser Thr Val Glu 20202025 2030 Thr Ser Val Ser Cys Phe Ser Lys Leu Ser Ala Ala Val Thr ThrAla 2035 2040 2045 Ser Gln Ala Glu Ala Leu Leu Ala Ile Leu Glu Gly TrpGlu Glu Leu 2050 2055 2060 Phe Glu Ala Lys Asn Ala Glu Leu Leu Pro SerAsn Glu Ala Thr Asp 2065 2070 2075 2080 Gln Gly Asn Asp Trp Gly Asp AspAsp Trp Asn Asp Gly Trp Glu Thr 2085 2090 2095 Leu Gln Glu Ser Glu ProVal Glu Lys Val Lys Lys Glu Cys Val Val 2100 2105 2110 Ser Ala His ProLeu His Ser Cys Trp Leu Asp Ile Phe Arg Lys Tyr 2115 2120 2125 Ile AlaLeu Ser Met Pro Glu Asn Val Leu Gln Leu Ile Asp Gly Ser 2130 2135 2140Leu Gln Lys Pro Glu Glu Val Ile Ile Glu Glu Thr Glu Ala Glu Ser 21452150 2155 2160 Leu Thr Gly Ile Leu Ala Arg Thr Asp Pro Phe Leu Ala LeuLys Ile 2165 2170 2175 Ser Leu Leu Leu Pro Tyr Lys Gln Ile Arg Ser GlnCys Leu Ser Val 2180 2185 2190 Val Glu Glu Gln Leu Lys Gln Glu Gly IlePro Glu Leu Ser Ser Gln 2195 2200 2205 Ser His His Glu Val Leu Leu LeuVal Ile Tyr Ser Gly Thr Leu Ser 2210 2215 2220 Thr Ile Ile Ser Asn AlaCys Tyr Gly Ser Val Phe Ser Phe Leu Cys 2225 2230 2235 2240 Tyr Leu IleGly Lys Leu Ser Arg Glu Phe Gln Glu Glu Arg Ile Thr 2245 2250 2255 GlnAla Asp Asn Arg Glu Ser Asn Ala Ser Ser Glu Ser Arg Phe Ile 2260 22652270 Ser Cys Phe Gly Gln Leu Met Phe Pro Cys Phe Val Ser Gly Leu Val2275 2280 2285 Lys Ala Asp Gln Gln Ile Leu Ala Gly Phe Leu Val Thr LysPhe Met 2290 2295 2300 His Ser Asn Pro Ser Leu Ser Leu Ile Asn Val AlaGlu Ala Ser Leu 2305 2310 2315 2320 Arg Arg Tyr Leu Asp Lys Gln Leu GluSer Leu Glu His Leu Glu Asp 2325 2330 2335 Ser Phe Ala Glu Ser Ser AspPhe Glu Thr Leu Lys Asn Thr Val Ser 2340 2345 2350 Ser Leu Arg Gly ThrSer Lys Glu Val Ile Arg Ser Ala Leu Ala Ser 2355 2360 2365 Leu Ser AsnCys Thr Asn Ser Arg 2370 2375 57 230 PRT Arabidopsis thaliana 57 Met AlaSer Pro Thr Ile Arg Thr Val Ala Ser Tyr Asn Thr His Leu 1 5 10 15 GluTyr Ser Val Asp Phe Phe Gly Asp Glu Phe Ile Val Thr Val Thr 20 25 30 TrpAsp Ser Ser Val Ile Ser Arg Trp Ile Arg Asn Val Leu Phe Asn 35 40 45 AsnArg Phe Ser Ser His Pro Leu Val Val Gly Val Gly Val Gln Trp 50 55 60 ThrPro Phe Ser Tyr Tyr Ser Asp Pro Arg Pro Asn Asn Tyr Tyr Ala 65 70 75 80Asp Pro Pro Pro Ile Arg Tyr Tyr Ser Asp Asn Pro Ala Asp Ile Leu 85 90 95Gln Leu Cys Val Gly Asn Arg Cys Leu Ile Ile Gln Leu Gly Tyr Cys 100 105110 Asp Gln Val Pro Asn Asn Leu Arg Ser Phe Leu Ala Asp Pro Glu Thr 115120 125 Thr Phe Val Gly Val Trp Asn Gly Gln Asp Ala Gly Lys Leu Ala Arg130 135 140 Cys Cys His Gln Leu Glu Ile Gly Glu Leu Leu Asp Ile Arg ArgTyr 145 150 155 160 Val Thr Asp Ser Trp Gly Arg Ser Met Arg Arg Ser SerPhe Glu Glu 165 170 175 Ile Val Glu Glu Cys Met Gly Tyr Gln Gly Val MetLeu Asp Pro Glu 180 185 190 Ile Ser Met Ser Asp Trp Thr Ala Tyr Asp LeuAsp Leu Asp Gln Ile 195 200 205 Leu Gln Ala Ser Leu Asp Ala Tyr Val CysHis Gln Leu Gly Val Trp 210 215 220 Thr Arg Leu Trp Glu Val 225 230

What is claimed is:
 1. An isolated polynucleotide comprising: (a) a nucleotide sequence encoding a polypeptide having RNaseD activity, wherein the amino acid sequence of the polypeptide and the amino acid sequence of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 46, or 48 have at least 80% sequence identity based on the Clustal alignment method, (b) the complement of the nucleotide sequence.
 2. The isolated polynucleotide of claim 1, wherein the amino acid sequence of the polypeptide and the amino acid sequence of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 46, or 48 have at least 85% sequence identity based on the Clustal alignment method.
 3. The isolated polynucleotide of claim 1, wherein the amino acid sequence of the polypeptide and the amino acid sequence of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, or 48 have at least 90% sequence identity based on the Clustal alignment method.
 4. The isolated polynucleotide of claim 1, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, or 48 have at least 95% sequence identity based on the Clustal alignment method.
 5. A chimeric gene comprising the polynucleotide of claim 1 operably linked to a regulatory sequence.
 6. A vector comprising the polynucleotide of claim
 1. 7. An isolated polynucleotide fragment comprising a nucleotide sequence containing at least 30 nucleotides, wherein the nucleotide sequence containing at least 30 nucleotides is comprised by the polynucleotide of claim
 1. 8. The fragment of claim 7, wherein the nucleotide sequence containing at least 30 nucleotides contains at least 40 nucleotides.
 9. The fragment of claim 7, wherein the nucleotide sequence containing at least 30 nucleotides contains at least 60 nucleotides.
 10. A method for transforming a cell comprising transforming a cell with the polynucleotide of claim
 1. 11. A cell comprising the chimeric gene of claim
 5. 12. A method for producing a transgenic plant comprising transforming a plant cell with the polynucleotide of claim 1 and regenerating a plant from the transformed plant cell.
 13. A plant comprising the chimeric gene of claim
 5. 14. A seed comprising the chimeric gene of claim
 5. 15. An isolated polypeptide having RNaseD activity, wherein the polypeptide comprises an amino acid sequence, wherein the amino acid sequence and the amino acid sequence of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 46, or 48 have at least 80% sequence identity based on the Clustal alignment method.
 16. The polypeptide of claim 15, wherein the amino acid sequence and the amino acid sequence of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 46, or 48 have at least 85% sequence identity based on the Clustal alignment method.
 17. The polypeptide of claim 15, wherein the amino acid sequence and the amino acid sequence of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, or 48 have at least 90% sequence identity based on the Clustal alignment method.
 18. The polypeptide of claim 15, wherein the amino acid sequence and the amino acid sequence of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, or 48 have at least 95% sequence identity based on the Clustal alignment method.
 19. A method for evaluating a compound for its ability to inhibit the activity of a polypeptide having RNaseD activity, wherein the method comprises: (a) transforming a host cell with a chimeric gene comprising a nucleotide sequence encoding a polypeptide having RNaseD activity, wherein the nucleotide sequence is operably linked to a regulatory sequence, (b) growing the transformed host cell under conditions that are suitable for expression of the chimeric gene, wherein the expression results in the production of the polypeptide in the transformed host cell, (c) optionally purifying the produced polypeptide, (d) treating the produced polypeptide with a compound, (e) comparing the activity of the treated polypeptide to the activity of an untreated polypeptide having RNaseD activity. 